RS7 antibodies

ABSTRACT

This invention relates to monovalent and multivalent, monospecific binding proteins and to multivalent, multispecific binding proteins. One embodiment of these binding proteins has one or more binding sites where each binding site binds with a target antigen or an epitope on a target antigen. Another embodiment of these binding proteins has two or more binding sites where each binding site has affinity towards different epitopes on a target antigen or has affinity towards either a target antigen or a hapten. The present invention further relates to recombinant vectors useful for the expression of these functional binding proteins in a host. More specifically, the present invention relates to the tumor-associated antigen binding protein designated RS7, and other EGP-1 binding-proteins. The invention further relates to humanized, human and chimeric RS7 antigen binding proteins, and the use of such binding proteins in diagnosis and therapy.

This application is a continuation of U.S. patent application Ser. No.11/745,896 (now issued U.S. Pat. No. 7,517,964) filed on May 8, 2007which is a divisional of U.S. patent application Ser. No. 10/377,121 nowissued U.S. Pat. No. 7,238,785 filed on Mar. 3, 2003, entitled “RS7Antibodies” which claims priority to U.S. Provisional Application No.60/360,299 expired, filed Mar. 1, 2002, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to monovalent and multivalent, monospecificbinding proteins and to multivalent, multispecific binding proteins. Oneembodiment of these binding proteins has one or more binding sites whereeach binding site binds with a target antigen or an epitope on a targetantigen. Another embodiment of these binding proteins has two or morebinding sites where each binding site has affinity towards differentepitopes on a target antigen or has affinity towards either a targetantigen or a hapten. The present invention further relates torecombinant vectors useful for the expression of these functionalbinding proteins in a host. More specifically, the present inventionrelates to the tumor-associated antigen binding protein designated RS7.The invention further relates to humanized RS7 antigen binding proteins,and the use of such binding proteins in diagnosis and therapy.

BACKGROUND OF THE INVENTION

Man-made binding proteins, in particular monoclonal antibodies andengineered antibodies or antibody fragments, have been tested widely andshown to be of value in detection and treatment of various humandisorders, including cancers, autoimmune diseases, infectious diseases,inflammatory diseases, and cardiovascular diseases (Filpula and McGuire,Exp. Opin. Ther. Patents (1999) 9: 231-245). For example, antibodieslabeled with radioactive isotopes have been tested to visualize tumorsafter injection to a patient using detectors available in the art. Theclinical utility of an antibody or an antibody-derived agent isprimarily dependent on its ability to bind to a specific targetedantigen. Selectivity is valuable for delivering a diagnostic ortherapeutic agent, such as isotopes, drugs, toxins, cytokines, hormones,growth factors, enzymes, conjugates, radionuclides, or metals, to atarget location during the detection and treatment phases of a humandisorder, particularly if the diagnostic or therapeutic agent is toxicto normal tissue in the body.

The potential limitations of antibody systems are discussed inGoldenberg, The American Journal of Medicine (1993) 94: 298-299. Theimportant parameters in the detection and treatment techniques are theamount of the injected dose specifically localized at the site(s) wheretarget cells are present and the uptake ratio, i.e. the ratio of theconcentration of specifically bound antibody to that of theradioactivity present in surrounding normal tissues. When an antibody isinjected into the blood stream, it passes through a number ofcompartments as it is metabolized and excreted. The antibody must beable to locate and bind to the target cell antigen while passing throughthe rest of the body. Factors that control antigen targeting includelocation, size, antigen density, antigen accessibility, cellularcomposition of pathologic tissue, and the pharmacokinetics of thetargeting antibodies. Other factors that specifically affect tumortargeting by antibodies include expression of the target antigens, bothin tumor and other tissues, and bone marrow toxicity resulting from theslow blood-clearance of the radiolabeled antibodies. The amount oftargeting antibodies accreted by the targeted tumor cells is influencedby the vascularization and barriers to antibody penetration of tumors,as well as intratumoral pressure. Non-specific uptake by non-targetorgans such as the liver, kidneys or bone-marrow is another potentiallimitation of the technique, especially for radioimmunotherapy, whereirradiation of the bone marrow often causes the dose-limiting toxicity.

One suggested approach, referred to as direct targeting, is a techniquedesigned to target specific antigens with antibodies carrying diagnosticor therapeutic radioisotopes. In the context of tumors, the directtargeting approach utilizes a radiolabeled anti-tumor monospecificantibody that recognizes the target tumor through its antigens. Thetechnique involves injecting the labeled monospecific antibody into thepatient and allowing the antibody to localize at the target tumor toobtain diagnostic or therapeutic benefits. The unbound antibody clearsthe body. This approach can be used to diagnose or treat additionalmammalian disorders.

Another suggested solution, referred to as the “Affinity EnhancementSystem” (AES), is a technique especially designed to overcomedeficiencies of tumor targeting by antibodies carrying diagnostic ortherapeutic radioisotopes (U.S. Pat. No. 5,256,395 (1993), Barbet etal., Cancer Biotherapy & Radiopharmaceuticals (1999) 14: 153-166). TheAES utilizes a radiolabeled hapten and an anti-tumor/anti-haptenbispecific binding protein that recognizes both the target tumor and theradioactive hapten. Haptens with higher valency and binding proteinswith higher specificity may also be utilized for this procedure. Thetechnique involves injecting the binding protein into the patient andallowing it to localize at the target tumor. After a sufficient amountof time for the unbound binding protein to clear from the blood stream,the radiolabeled hapten is administered. The hapten binds to theantibody-antigen complex located at the site of the target cell toobtain diagnostic or therapeutic benefits. The unbound hapten clears thebody. Barbet mentions the possibility that a bivalent hapten maycrosslink with a bispecific antibody, when the latter is bound to thetumor surface. As a result, the radiolabeled complex is more stable andstays at the tumor for a longer period of time. This system can be usedto diagnose or treat mammalian disorders.

There remains a need in the art for production of multivalent,monospecific binding proteins that are useful in a direct targetingsystem and for production of multivalent, multispecific binding proteinsthat are useful in an affinity enhancement system. Specifically, thereremains a need for a binding protein that exhibits enhanced uptake attargeted antigens, decreased concentration in the blood, and optimalprotection of normal tissues and cells from toxic pharmaceuticals.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amonospecific monoclonal antibody and fragments thereof that recognizes atumor-associated antigen, defined as epithelial glycoprotein-1 (EGP-1)by the murine MAb RS7-3G11 raised against human non-small-cell lungcarcinoma. The RS7 antigen has been designated as EGP-1 (epithelialglycoprotein-1) following the proposal of the 3^(rd) International IASLCWorkshop on Lung Tumor and Differentiation Antigens. At least oneepitope associated with EGP-1 is alternatively referred to as TROP2 inthe literature. In a preferred embodiment, the antibody or antibodyfragment of the present invention binds the same epitope as the murineRS7 antibody disclosed by Stein (infra) and other earlier studies.Alternatively, the antibody or fragment may bind an epitope distinctfrom the epitope that the murine RS7 antibody disclosed by Stein binds.In a preferred embodiment, the anti-EGP-1, or anti-TROP2 antibody orfragment thereof is a chimeric, humanized, or fully human RS7 antibodyor fragment thereof.

For example, contemplated in the present invention is a humanizedantibody or fragment thereof, wherein the complementarity determiningregions (CDRs) of the light chain variable region of the humanized RS7MAb comprises CDR1 comprising an amino acid sequence of KASQDVSIAVA (SEQID NO: 28); CDR2 comprising an amino acid sequence of SASYRYT (SEQ IDNO: 29); and CDR3 comprising an amino acid sequence of QQHYITPLT (SEQ IDNO: 30). Another embodiment of the present invention is a humanizedantibody or fragment thereof, wherein the CDRs of the heavy chainvariable region of the humanized RS7 MAb comprises CDR1 comprising anamino acid sequence of NYGMN (SEQ ID NO: 31); CDR2 comprising an aminoacid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 32) and CDR3 comprisingan amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 33). Also preferred,the humanized antibody or fragment thereof of comprises the CDRs of amurine RS7 MAb and the framework region (FR) of the light and heavychain variable regions of a human antibody, wherein the CDRs of thelight chain variable region of the humanized RS7 MAb comprises CDR1comprising an amino acid sequence of KASQDVSIAVA (SEQ ID NO: 28); CDR2comprising an amino acid sequence of SASYRYT (SEQ ID NO: 29); and CDR3comprising an amino acid sequence of QQHYITPLT (SEQ ID NO: 30); and theCDRs of the heavy chain variable region of the humanized RS7 MAbcomprises CDR1 comprising an amino acid sequence of NYGMN (SEQ ID NO:31); CDR2 comprising an amino acid sequence of WINTYTGEPTYTDDFKG (SEQ IDNO: 32) and CDR3 comprising an amino acid sequence of GGFGSSYWYFDV (SEQID NO: 33). Still preferred, the humanized antibody or fragment thereoffurther comprises the FRs of the light and heavy chain constant regionsof a human antibody.

In a preferred embodiment, the humanized RS7 antibody or fragmentcomprises a FR of a light and/or heavy chain that comprises at least oneamino acid substituted by an amino acid residue found at a correspondinglocation in the RS7 murine antibody. For example, at least one of thesubstituted amino acids is preferably at a location selected from thegroup consisting of residue 38, 46, 68 and 91 of the murine heavy chainvariable region of FIG. 3B, and/or at least one of the substituted aminoacids is preferably at a location selected from the group consisting ofresidue 20, 85 and 100 of the murine light chain variable region of FIG.3A.

Also described in the present invention is an antibody fission proteinor fragment thereof that comprises at least two anti-EGP-1 MAb orfragments thereof, wherein the MAb or fragments thereof are selectedfrom the anti-EGP-1 MAb or fragments thereof of the present invention.In a related vein, the antibody fusion protein or fragment thereofcomprises at least one first anti-EGP-1 MAb or fragment thereof of anyof the anti-EGP-1 antibodies of the present invention and at least onesecond MAb or fragment thereof, other than the anti-EGP antibodies orfragment thereof in the present invention. For example, the secondantibody or fragment thereof may be a carcinoma-associated antibody orfragment thereof. Another preferred embodiment is a fusion protein orfragment thereof that comprises two different epitope-binding anti-EGP-1antibodies or fragments thereof.

It is one object of this invention to provide a multispecific antibodyand fragments thereof that recognize more than one epitope on the RS7antigen or that has affinity for the RS7 antigen and for a haptenmolecule. The latter binding protein is useful for pretargeting a targetantigen. Accordingly, a method of delivering a diagnostic agent, atherapeutic agent, or a combination thereof to a target, comprising: (i)administering to a subject a multivalent, multispecific MAb, or fragmentthereof (ii) waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and (iii)administering to said subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to abinding site of said antibody, is also described.

It is a further object of this invention to provide a method ofdelivering a diagnostic or therapeutic agent to a targeted disease thatexpresses EGP-1 antigen. For example, a method of delivering adiagnostic or therapeutic agent, or a combination thereof, to a targetcomprising (i) providing a composition that comprises an anti-EGP-1antibody or fragment thereof bound to at least one therapeutic and/ordiagnostic agent and (ii) administering to a subject in need thereofsaid composition, is described. Preferably, the diagnostic ortherapeutic agent is selected from the group consisting of an isotope,drug, toxin, immuno, modulator, hormone, enzyme, growth factor,radionuclide, metal, contrast agent, and detecting agent.

In another embodiment of the present invention, the method fordelivering a diagnostic agent, a therapeutic agent, or a combinationthereof to a target comprises (i) administering to a subject amultivalent, multispecific antibody or fragment comprising one or moreantigen-binding sites having affinity toward an EGP-1 target antigen andone or more hapten binding sites having an affinity toward a haptenmolecule, (ii) waiting a sufficient amount of time for an amount of thenon-binding antibody or fragment to clear a subject's blood stream, and(iii) administering to said subject a hapten comprising a diagnosticagent, a therapeutic agent, or a combination thereof.

Another object of the present invention to provide a cancer celltargeting diagnostic or therapeutic conjugate that comprises ananti-EGP-1 MAb or fragment thereof or an antibody fusion protein orfragment thereof of any one of antibodies of the present invention andwherein the anti-EGP-1 antibody or fragment thereof is bound to at leastone diagnostic or therapeutic agent. A suitable therapeutic agent is adrug that possesses the pharmaceutical property selected from the groupconsisting of an antimitotic, alkylating, antimetabolite,antiangiogenic, apoptotic, alkaloid antibiotic, and combinationsthereof. Also preferred is a therapeutic agent selected from the groupconsisting of a nitrogen mustard, ethylenimine derivative, alkylsulfonate, nitrosourea, triazene, folic acid analog, anthracycline,taxane, COX-2 inhibitor, tyrosine kinase inhibitor, pyrimidine analog,purine analog, antibiotic, enzyme, epipodophyllotoxin, platinumcoordination complex, vinca alkaloid, substituted urea, methyl hydrazinederivative, adrenocortical suppressant, antagonist, endostatin taxol,camptothecins, doxorubicin, doxorubicin analog, and a combinationthereof. Preferably, the diagnostic agent is selected from the groupconsisting of a photoactive radionuclide, preferably between 25 and 4000keV, and a contrast agent.

In a preferred embodiment, a DNA sequence comprising a nucleic acidencoding a MAb or fragment that contains a anti-EGP-1 MAb or fragmentthereof of the present invention; an antibody fusion protein or fragmentthereof containing at least two of said MAbs or fragments thereof; anantibody fusion protein or fragment thereof containing at least onefirst anti-EGP-1 MAb or fragment thereof containing the MAb or fragmentthereof of the anti-EGP-1 antibodies and fragments of the presentinvention and at least one second MAb or fragment thereof, other thanthe anti-EGP-1 MAb or fragment thereof described herein; or an antibodyfusion protein or fragment thereof comprising at least one first MAb orfragment thereof comprising said MAb or fragment thereof of any of theantibodies described herein and at least one second MAb or fragmentthereof, other than the MAb or fragment thereof of any one of theantibodies described herein, wherein the second MAb is reactive with anantigen selected from the group consisting of EGP-2, WC 1-4, A33, CSAp,CEA, Le(y), Tn, Tag-72, PSMA, PSA, EGFR, HER2/neu, AFP, HCG, HCG-beta,ferritin, PAP, PLAP, EGP-2, histone, cytokeratin, Tenascin, CanAg,kidney cancer G 250, VGFR1, VGFR2, P4-antigen, oncogene products, or acombination thereof. The second MAb may instead be reactive withvascular endothelial antigens associated with tumors, such as VEGF(vascular endothelial growth factor) and P1GF (placenta growth factor).Selection of the second antibody is dependent on tumor cell type. Forexample, anti-PSMA or anti-PSA antibodies may be used for treating ordiagnosing prostate cancer, anti-CEA or anti-MUC1, MUC2, MUC3 and MUC4antibodies for breast, ovarian, lung, and colon cancer, EGFR for colonand head and neck cancers, anti-CSAp antibodies for colon and ovariancancer, and anti-HER/neu for breast, ovarian and other cancers. Theseare merely given as examples, and are not intended to be limiting.Expression vectors and host cells containing this DNA sequence are alsopreferred embodiments of the present invention.

Also provided herein are methods for diagnosing and treating amalignancy. For example, a method for diagnosing or treating cancer,comprises (i) administering to a subject in need thereof a multivalent,multispecific antibody or fragment comprising one or moreantigen-binding sites having affinity toward an EGP-1 target antigen andone or more hapten binding sites having an affinity toward a haptenmolecule; (ii) waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and (iii)administering to said subject a hapten comprising a diagnostic agent, atherapeutic agent, or a combination thereof, that binds to a bindingsite of said antibody.

Likewise, the methods for diagnosing and treating a malignancy maycomprise administering a therapeutically effective amount of ananti-EGP-1 fusion protein or fragment thereof or a therapeutic conjugatecomprising a EGP-1 MAb or fragment thereof, wherein the EGP-1 MAb orfragment thereof or antibody fusion protein or fragment thereof is boundto at least one therapeutic agent in a pharmaceutically suitableexcipient. In a related vein, naked anti-EGP-1 antibodies and fragmentsthereof, including naked anti-EGP-1 fusion proteins and fragmentsthereof, can also be used for treating a malignancy. Naked anti-EGP-1antibodies may be used for in vitro diagnosis of a malignancy, forexample with immunoassays or immunohistochemistry, but not for in vivodiagnosis, unless this involves a pretargeting technology, such as AES.Labeled EGP-1 antibodies, however, may be used for in vivo diagnosis andtreatment of a malignancy. For example, described herein is a method oftreating a cancer cell in a subject comprising (i) administering to asubject a therapeutically effective amount of a composition containingan anti-EGP-1 MAb or fragment thereof or an antibody fusion protein orfragment thereof (ii) formulating the EGP-1 MAb or fragment thereof orantibody fission protein or fragment thereof in a pharmaceuticallysuitable excipient. Similarly, combinations of naked MAbs and fragmentsthereof with conjugated MAbs or fragments thereof or fusion proteins orfragments thereof for diagnosis and treatment are also contemplated inthe instant invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of mRS7, cAb-Vκ#23 (cRS7), and cAb-Vκ#1 incompetitive binding assays. Varying concentrations of competing Abs wereused to compete with the binding of a constant amount of biotinylatedmRS7 antibody. Results indicate that the Vκ#1 light chain does not bindthe RS7 antigen.

FIG. 2 shows the DNA and amino acid sequences encoding (A) RS7 Vκ (SEQID NOS: 1-2, respectively, in order of appearance) cloned by 5′ RACE and(B) RS7 VH (SEQ ID NOS: 3-4, respectively, in order of appearance)cloned by RT-PCR. The putative CDR regions are underlined and indicated.Nucleotide residues are numbered sequentially. Kabat's Ig moleculenumbering is used for amino acid residues. In (B), the numbering for theresidues with a letter (on top) is the number of preceding residues plusthe letter, e.g., the number for T following N52 is 52A; the numbers forN, N and L following 182 are 82A, 82B and 82C, respectively.

FIG. 3 shows the amino acid sequence alignment of (A) human SA-IA'cl(SEQ ID NO: 5), murine RS7 (SEQ ID NO: 2), and hRS7 (SEQ ID NO: 7) Vκchains and (B) human RF-TS3 (SEQ ID NO: 8), murine RS7 (SEQ ID NO: 4,SEQ ID NO: 9), and hRS7 (SEQ ID NO: 10, SEQ ID NO: 27) V_(H) chains. In(A), dots indicate the residues in RS7 are identical to thecorresponding residues in SA-1A'cl. Dashes represent gaps introduced toaid the alignment. Boxed represent the CDR regions. Both N- andC-terminal residues (underlined) of hRS7 are fixed by the staging vectorused. Therefore, the corresponding terminal residues of RS7 are notcompared with that of the human sequence. Kabat's numbering scheme isused. In (B), dots indicate the residues in RS7 are identical to thecorresponding residues in RF-TS3. Dashes represent gaps introduced toaid the alignment. Boxed represent the CDR regions. Both N- andC-terminal residues (underlined) of hRS7 are fixed by the staging vectorused. Therefore, the corresponding terminal residues of RS7 are notcompared with that of the human VH sequence.

FIG. 4 shows the DNA and amino acid sequences for (A) humanized RS7 Vκ(SEQ ID NO: 11-12, respectively, in order of appearance) and (B)humanized RS7 V_(H) (SEQ ID NO: 13-14, respectively, in order ofappearance). The bold and underlined sections of the amino acidsequences indicate the CDRs as defined by the Kabat numbering scheme.

FIG. 5 shows the (A) light chain cDNA (SEQ ID NO: 15) and amino acid(SEQ ID NO: 16) sequences for humanized RS7 Vκ and (B) heavy chain cDNA(SEQ ID NO: 17) and amino acid (SEQ ID NO: 18) sequences for humanizedRS7 V_(H). The underlined sections of the amino acid sequences indicatethe leader peptide sequence for secretion. “*” indicates the stop codon.

FIG. 6 shows a comparison of mRS7, cRS7, and hRS7 in competitive bindingassays. Varying concentrations of competing Abs were used to competewith the binding of a constant amount of Biotinylated RS7 to the Agcoated in 96-well ELISA plates. hRS7 showed comparable blocking activityas that of RS7 and cRS7.

FIG. 7 indicates the structure of the residualizing moieties IMP-R4,IMP-R5 and IMP-R8.

FIG. 8 is a bar graph of dosimetry due to radioiodinated hRS7 in theMDA-MB-468 tumor model.

FIGS. 9A-9C show tumor growth controls, as plots of tumor volumes (cm³)in Y-axis versus days post-treatment in X-axis, in individual NIH Swissnude mice (female) subcutaneously carrying MDA-MB-468 human breastcarcinoma xenografts, and which were either treated with 0.2 mCi ofconventionally ¹³¹I-radioiodinated hRS7, ¹³¹I-hRS7 (FIG. 9C), or treatedwith 0.175 mCi of hRS7 antibody radioiodinated with ¹³¹I-IMP-R4 which isa residualizing form of ¹³¹I, ¹³¹I-IMP-R4-hR4S7 (FIG. 9B), or wereuntreated (FIG. 9A). Each line in each of these graphs corresponds totumor growth in a single mouse. FIG. 9D is a composite of the tumorgrowth controls in the different groups, and represents mean tumorvolumes, as a function of time in days, in animals that were treatedwith 0.175 mCi of ¹³¹I-IMP-R4-hRS7 (solid square) or were treated with0.2 mCi of ¹³¹I-hRS7 (open triangle) or were untreated (solid diamond).Error bar represents standard deviation. FIG. 9E is a differentrepresentation of tumor growth control vs. time plots, showing meanrelative tumor volumes as a function of time in various groups with meantumor volume at the start of therapy taken as 100. Otherwise, the legendis the same as for 9D. Error bar represents standard deviation.

FIGS. 10A and 10B depict determinations of myelotoxicities of treatmentswith radioiodinated hRS7 in MDA-MB-468 human tumor xenograft-bearingSwiss nude mice (female). FIG. 10A shows the data for treatment with0.175 mCi of hRS7 radioiodinated with a residualizing form of ¹³¹I (i.e.¹³¹I-IMP-R4-hRS7); and FIG. 10B shows data for treatment with 0.2 mCi ofhRS7 conventionally radioiodinated with ¹³¹I (i.e. ¹³¹I-hRS7). In bothfigures, mean white blood cell counts (solid diamond), mean lymphocytecounts (solid square), mean monocyte counts (open triangle), and meanneutrophil counts (‘X’), expressed as percentage of respective meanvalues in untreated control animals, are shown as a function of time inweeks.

FIG. 11 is a graph demonstrating relative mean tumor volumes (MTV).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise specified, “a” or “an” means “one or more.”

An RS7 antibody (previously designated RS7-3G11) is a murine IgG.sub.1raised against a crude membrane preparation of a human primary squamouscell carcinoma from the lung. See Stein et al., Cancer Res. 50: 1330(1990), which is fully incorporated by reference. The RS7 antibodyrecognizes a tumor-associated antigen, which was defined by the murineMAb RS7-3G11 raised against human non-small-cell lung carcinoma. Steinet al. discloses that the RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. Stein et al., Int. J. CancerSupp. 8:98-102 (1994). See also, Basu et al., Int. J. Cancer 52:472-479(1995). The antigen has been designated as EGP-1 (epithelialglycoprotein-1) following the proposal of the 3.sup.rd InternationalIASLC Workshop on Lung Tumor and Differentiation Antigens. See, forexample DeLeij et al., Int. J. Cancer Supp., 8:60-63 (1994).Accordingly, as described herein, the RS7 and EGP-1 antigens aresynonymous. The EGP-1 antigen is also referred to as TROP2 in theliterature, but there may be multiple epitopes of both EGP-1 and TROP2.

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue. (Stein et al., (1990), supra). The RS7antibody is reactive with an EGP-1 glycoprotein, which can be rapidlyinternalized. EGP-1 is expressed primarily by carcinomas such ascarcinomas of the lung, stomach, urinary bladder, breast, ovary, uterus,and prostate. Localization and therapy studies using radiolabeled murineRS7 MAb in animal models have demonstrated tumor targeting andtherapeutic efficacy (Stein et al., (1990), supra. Stein et al., (1991),supra).

A more recent study has demonstrated strong RS7 staining in tumors fromthe lung, breast, bladder, ovary, uterus, stomach, and prostate. SeeStein et al., Int. J. Cancer 55: 938 (1993), which is fully incorporatedby reference. Moreover, the lung cancer cases in this study comprisedboth squamous cell carcinomas and adenocarcinomas. Id. Both cell typesstained strongly, indicating that the RS7 antibody does not distinguishbetween histologic classes of non-small-cell carcinoma of the lung.

As discussed supra, the RS7 MAb is rapidly internalized into targetcells (Stein et al.(1993), supra). The internalization rate constant forRS7 MAb is intermediate between the internalization rate constants oftwo other rapidly internalizing MAbs, which have been demonstrated to beuseful for immunotoxin production. Id. It is well documented that theinternalization of immunotoxin conjugates is an absolute requirement foranti-tumor activity. (Pastan et al., Cell 47:641 (1986)).Internalization of drug immunoconjugates also has been described as amajor factor in anti-tumor efficacy. (Yang et al., Proc. Nat'l Acad.Sci. USA 85: 1189 (1988)). Therefore, the RS7 antigen may be animportant target for those types of immunotherapy that requireinternalization of the therapeutic agent.

Thus, studies with the RS7 MAb indicate that the antibody exhibitsseveral important properties, which make it a candidate for clinicaldiagnostic and therapeutic applications. Since the RS7 antigen providesa useful target for diagnosis and therapy, it is desirable to obtain aMAb that recognizes an epitope of the RS7 antigen. Moreover, theavailability of chimeric, humanized and human RS7 antibodies isessential for the development of a double-determinant enzyme-linkedimmunosorbent assay (ELISA), which is desirable for detecting the RS7antigen in clinical samples, and essential for in vivo applications inhumans.

To this end, the present invention describes chimeric, humanized andhuman antibodies and fragments thereof that bind the RS7 antigen and canbe used for diagnostic and therapeutic methods. Humanized antibodies andantibody fragments are described in Provisional U.S. Application titled“Anti-CD20 Antibodies And Fusion Proteins Thereof And Methods Of Use”,U.S. Provisional Application No. 60/356,132, filed Feb. 14, 2002,(expired), and U.S. Provisional Application No. 60/416,232, filed Oct.7, 2002, (expired), both now U.S. application Ser. No. 10/366,709, filedFeb. 4, 2003 (PGP No. US 2003-0219433-A1, now issued U.S. Pat. No.7,151,164); hMN-14 antibodies, such as those disclosed in U.S. Pat. No.5,874,540, which is a Class III anti-carcinoembryonic antigen antibody(anti-CEA antibody); Mu-9 antibodies, such as those described in U.S.application Ser. No. 10/116,116 (now U.S. Pat. No. 7,387,772), filedApr. 5, 2002, titled “Chimeric, Human And Humanized Anti-CSAP MonoclonalAntibodies;” AFP antibodies, such as those described in U.S. ProvisionalApplication No. 60/399,707, filed Aug. 1, 2002, titled“Alpha-Fetoprotein IMMU31 Antibodies And Fusion Proteins And Methods OfUse Thereof,” (expired), now U.S. application Ser. No. 10/631,722, filedAug. 1, 2003 (PGP No. US 2004-0235065 A1, now issued U.S. Pat. No.7,300,655); PAM4 antibodies, such as those described in Provisional U.S.Application No. 60/388,313, filed Jun. 14, 2002 (expired), titled“Monoclonal Antibody cPAM4, now U.S. application Ser. No. 10/461,878filed Jun. 16, 2003 (PGP No. 2004/0057902, now issued U.S. Pat. No.7,238,786)”; RS7 antibodies, such as those described in U.S. ProvisionalApplication No. 60/360,229, filed Mar. 1, 2002 (expired), from whichthis application claims priority; and CD22 antibodies, such as thosedisclosed in U.S. Pat. Nos. 5,789,554 and 6,187,287 and U.S. applicationSer. Nos. 09/741,843 (PGP No. US-2002-0102254-A1, now abandoned) andSer. No. 09/988,013 (PGP No. US 2003-0103979-A1), all of which areincorporated herein by reference in their entirety. A chimeric antibodyas disclosed herein is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species. A humanizedantibody is a recombinant protein in which the CDRs from an antibody ofone species, e.g., a rodent antibody, are transferred from the heavy andvariable chains of the rodent antibody into human heavy and lightvariable domains.

In a preferred embodiment, the RS7 antibody is humanized. Becausenon-human monoclonal antibodies can be recognized by the human host as aforeign protein, and repeated injections can lead to harmfulhypersensitivity reactions, humanization of a murine RS7 sequences canreduce the adverse immune response that patients may experience. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response. Another embodiment of the presentinvention is an anti-EGF-1 antibody or fragment thereof that is asubhuman primate anti-EGP-1 antibody, murine monoclonal anti-EGP-1antibody (restricted to veterinary applications), chimeric anti-EGP-1antibody, human anti-EGP-1 antibody, and humanized anti-EGP-1 antibody.Preferably, the chimeric, human and humanized anti-EGP-1 antibodycomprises constant and hinge regions of a human IgG1. Also preferred,some human residues in the framework regions of the humanized RS7antibody or fragments thereof are replaced by their murine counterparts.It is also preferred that a combination of framework sequences from 2different human antibodies are used for V_(H). The constant domains ofthe antibody molecule are derived from those of a human antibody.

Another preferred embodiment of the present invention is a human RS7antibody. A human antibody is an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

The antibody and fragments thereof of the present invention ispreferably raised against a crude membrane preparation from a humanprimary squamous cell carcinoma of the lung. Also preferred, the RS7antibody and fragments thereof is raised against a membrane preparationof viable cells from a human ovarian carcinoma cell line. Stillpreferred, the RS7 antigen is provided by viable Colo 316 cells. In arelated vein, the RS7 antibody can be obtained using a substantiallypure preparation of the RS7 antigen. A substantially pure protein is aprotein that is essentially free from contaminating cellular components,which are associated with the protein in nature. As described herein,the term “RS7 antibody” also includes chimeric, human and humanized RS7antibodies.

Preparation of Chimeric, Humanized and Human RS7 Antibodies

Monoclonal antibodies to specific antigens may be obtained by methodsknown to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CurrentProtocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, RS7 antigen MAbs, such as RS7,can be obtained by injecting mice with a composition comprising the RS7antigen, verifying the presence of antibody production by removing aserum sample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to RS7antigen, culturing the clones that produce antibodies to RS7 antigen,and isolating RS7 antibodies from the hybridoma cultures.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

A human antibody of the present invention, i.e., human EGP-1 MAbs orother human antibodies, such as anti-EGP-2, MUC1-4, CEA, CC49, CSAp,PSMA, PSA, EGFR, A33 and HER2/neu MAbs for combination therapy withhumanized, chimeric or human RS7 antibodies, can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425, which are incorporated intheir entirety by reference. A human antibody of the present inventionthat can be used for combination therapy may also be reactive with anantigen selected from the group consisting of Le(y), Tn, Tag-72, AFP,HCG, HCG-beta, ferritin, PAP, EGP-2, histone, cytokeratin, Tenascin,CanAg, kidney cancer G 250, VGFR1, VGFR2, or a combination thereof. Forexample, a human antibody can be recovered from a transgenic mousepossessing human immunoglobulin loci. The mouse humoral immune system ishumanized by inactivating the endogenous immunoglobulin genes andintroducing human immunoglobulin loci. The human immunoglobulin loci areexceedingly complex and comprise a large number of discrete segmentswhich together occupy almost 0.2% of the human genome. To ensure thattransgenic mice are capable of producing adequate repertoires ofantibodies, large portions of human heavy- and light-chain loci must beintroduced into the mouse genome. This is accomplished in a stepwiseprocess beginning with the formation of yeast artificial chromosomes(YACs) containing either human heavy- or light-chain immunoglobulin lociin germline configuration. Since each insert is approximately 1 Mb insize, YAC construction requires homologous recombination of overlappingfragments of the immunoglobulin loci. The two YACs, one containing theheavy-chain loci and one containing the light-chain loci, are introducedseparately into mice via fusion of YAC-containing yeast spheroblastswith mouse embryonic stem cells. Embryonic stem cell clones are thenmicroinjected into mouse blastocysts. Resulting chimeric males arescreened for their ability to transmit the YAC through their germlineand are bred with mice deficient in murine antibody production. Breedingthe two transgenic strains, one containing the human heavy-chain lociand the other containing the human light-chain loci, creates progeny,which produce human antibodies in response to immunization.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat.'l Acad. Sci. USA 86: 3833 (1989), which is incorporated byreference in its entirety. Techniques for producing humanized MAbs aredescribed, for example, by Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Singer et al., J. Immun. 150: 2844 (1992), Mountain et al.Biotechnol. Genet. Eng. Rev. 10:1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is hereby incorporated by reference.

In general, the Vκ (variable light chain) and V_(H) (variable heavychain) sequences for RS7 antibodies can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the VH and Vκ genes of the MAb RS7 were clonedby PCR amplification from the hybridoma cells by RT-PCR and 5′ RACE,respectively, and their sequences determined by DNA sequencing. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)) which isincorporated by reference. Based on the V gene sequences, a humanizedRS7 antibody can then be designed and constructed as described by Leunget al. (Mol. Immunol., 32: 1413 (1995)), which is incorporated byreference. cDNA can be prepared from any known hybridoma line ortransfected cell line producing a murine or chimeric RS7 antibody bygeneral molecular cloning techniques (Sambrook et al., MolecularCloning, A laboratory manual, 2^(nd) Ed (1989)). In a preferredembodiment, the RS7 hybridoma line is used. The Vκ sequence for the mAbmay be amplified using the primers VK1BACK and VK1FOR (Orlandi et al.,1989) or the extended primer set described by Leung et al.(BioTechniques, 15: 286 (1993)), which is incorporated by reference,while V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989 above), or the primers annealing tothe constant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)), which is incorporated by reference. The PCR reactionmixtures containing 10 μl of the first strand cDNA product, 10 μl of10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM ofeach dNTP, 200 nM of the primers, and 5 units of Taq DNA polymerase(Perkin Elmer Cetus) can be subjected to 30 cycles of PCR. Each PCRcycle preferably consists of denaturation at 94° C. for 1 min, annealingat 50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min.Amplified Vκ and V_(H) fragments can be purified on 2% agarose (BioRad,Richmond, Calif.). Similarly, the humanized V genes can be constructedby a combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the Vκ PCR products. PCR products for V_(H) can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)), which is incorporated by reference.

The DNA sequences described herein are to be taken as including allalleles, mutants and variants thereof, whether occurring naturally orinduced.

The expression cassettes containing the Vκ and VH, together with thepromoter and signal peptide sequences can be excised from VKpBR andVHpBS, respectively, by double restriction digestion as HindIII-BamHIfragments. The Vκ and VH expression cassettes can then be ligated intoappropriate expression vectors, such as pKh and pG1g, respectively(Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can beco-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14 (ATCC,VA), colonies selected for hygromycin resistance, and supernatant fluidsmonitored for production of a chimeric or humanized RS7 MAb by, forexample, an ELISA assay, as described below. Alternately, the Vκ and VHexpression cassettes can be assembled in the modified staging vectors,VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHI fragments,respectively, and subcloned into a single expression vector, such aspdHL2, as described by Gilles et al. (J. Immunol. Methods 125:191 (1989)and also shown in Losman et al., Cancer, 80:2660 (1997)) for theexpression in Sp2/0-Ag14 cells. Another vector that is useful in thepresent invention is the GS vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNSO cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(11), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992) which is incorporated by reference. Following transfection,cells may be grown in 96-well microtiter plates in complete HSFM medium(Life Technologies, Inc., Grand Island, N.Y.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 units/ml of hygromycin. Colonies typically emerge2-3 weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Suitable host cells include microbial or mammalian host cells. Apreferred host is the human cell line, PER.C6, which was developed forproduction of MAbs, and other fusion proteins. Accordingly, a preferredembodiment of the present invention is a host cell comprising a DNAsequence encoding and anti-EGP-1 MAb, conjugate, fusion protein orfragments thereof. PER.C6 cells (WO 97/00326) were generated bytransfection of primary human embryonic retina cells, using a plasmidthat contained the Adserotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5nucleotides 459-3510) under the control of the human phosphoglyceratekinase (PGK) promoter. E1A and E1B are adenovirus early gene activationprotein 1A and 1B, respectively. The methods and compositions areparticularly useful for generating stable expression of humanrecombinant proteins of interest that are modified post-translationally,e.g. by glycosylation. Several features make PER.C6 particularly usefulas a host for recombinant protein production, such as PER.C6 is a fullycharacterized human cell line and it was developed in compliance withgood laboratory practices. Moreover, PER.C6 can be grown as a suspensionculture in defined serum-free medium devoid of any human- oranimal-derived proteins and its growth is compatible with rollerbottles, shaker flasks, spinner flasks and bioreactors with doublingtimes of about 35 hrs. Finally, the presence of E1A causes an upregulation of expression of genes that are under the control of the CMVenhancer/promoter and the presence of E13 prevents p53-dependentapoptosis possibly enhanced through over expression of the recombinanttransgene. In one embodiment, the cell is capable of producing 2 to200-fold more recombinant protein and/or proteinaceous substance thanconventional mammalian cell lines.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (˜100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 hr at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch) are added to the wells, (100 μl of antibody stockdiluted×10⁴, supplemented with the unconjugated antibody to a finalconcentration of 1.0 μg/ml). Following an incubation of 1 h, the platesare washed, typically three times. A reaction solution, [100 μl,containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μm of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

The nucleotide sequences of the primers used to prepare the RS7antibodies are listed in Example 2, below. In a preferred embodiment, ahumanized RS7 antibody or antibody fragment comprises thecomplementarity-determining regions (CDRs) of a murine RS7 MAb and theframework (FR) regions of the light and heavy chain variable regions ofa human antibody and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized RS7 comprises CDR1 comprising an amino acid sequence ofKASQDVSIAVA (SEQ ID NO: 28); CDR2 comprising an amino acid sequence ofSASYRYT (SEQ ID NO: 29); and CDR3 comprising an amino acid sequence ofQQHYITPLT (SEQ ID NO: 30); and the CDRs of the heavy chain variableregion of the humanized RS7 MAb comprises CDR1 comprising an amino acidsequence of NYGMN (SEQ ID NO: 31); CDR2 comprising an amino acidsequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 32) and CDR3 comprising anamino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 33). Also preferred, theFRs of the light and heavy chain variable regions of the humanizedantibody comprise at least one amino acid substituted from saidcorresponding FRs of the murine RS7 MAb.

RS7 MAbs can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992).

RS7 MAbs can be characterized by a variety of techniques that arewell-known to those of skill in the art. For example, the ability of anRS7 MAb to bind to the RS7 antigen can be verified using an indirectimmunofluorescence assay, flow cytometry analysis, or Western analysis.

Production of RS7 Antibody Fragments

The present invention contemplates the use of fragments of RS7 and hRS7antibodies. Antibody fragments, which recognize specific epitopes, canbe generated by known techniques. The antibody fragments are antigenbinding portions of an antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv andthe like. Other antibody fragments include, but are not limited to: theF(ab)′₂ fragments which can be produced by pepsin digestion of theantibody molecule and the Fab′ fragments, which can be generated byreducing disulfide bridges of the F(ab)′₂ fragments. These methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein, which patents areincorporated herein in their entireties by reference. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods in Enzymology, Vol. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity. The present invention encompasses antibodies and antibodyfragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” Faseb, Vol. 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TibTech, Vol. 9: 132-137(1991). These references are incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in Methods in Enzymology, Vol. 1,page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MonoclonalAntibodies: Production, Engineering and Clinical Application, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MonoclonalAntibodies: Principles and Applications, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Production of Chimeric, Humanized and Human RS7 Antibody Fusion Proteins

Antibody fusion proteins and fragments thereof can be prepared by avariety of conventional procedures, ranging from glutaraldehyde linkageto more specific linkages between functional groups. The antibodiesand/or antibody fragments are preferably covalently bound to oneanother, directly or through a linker moiety, through one or morefunctional groups on the antibody or fragment, e.g., amine, carboxyl,phenyl, thiol, or hydroxyl groups. Various conventional linkers inaddition to glutaraldehyde can be used, e.g., disiocyanates,diiosothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxysuccinimide esters, and the like.

A simple method to produce chimeric, humanized and human RS7 antibodyfusion proteins is to mix the antibodies or fragments in the presence ofglutaraldehyde to form an antibody fusion protein. The initial Schiffbase linkages can be stabilized, e.g., by borohydride reduction tosecondary amines. A diiosothiocyanate or carbodiimide can be used inplace of glutaraldehyde as a non-site-specific linker. Antibody fusionproteins are expected to have a greater binding specificity than MAbs,since the fusion proteins comprise moieties that bind to at least twoepitopes of the RS7 antigen. Thus, antibody fusion proteins arc thepreferred form of RS7 antigen binding protein for therapy.

In the present context, an antibody fusion protein comprises at leasttwo chimeric, humanized or human RS7 MAbs, or fragments thereof, whereinat least two of the MAbs or fragments bind to different epitopes of theRS7 antigen or against an RS7 epitope and that of a totally differentantigen. For example, a bispecific RS7 antibody fusion protein maycomprise a CEA antibody or fragment thereof and the RS7 MAb or fragmentthereof. Such a bispecific RS7 antibody fusion protein can be prepared,for example, by obtaining an F(ab′)₂ fragment from CEA as describedabove. The interchain disulfide bridges of the antibody F(ab′)₂ fragmentare gently reduced with cysteine, taking care to avoid light-heavy chainlinkage, to form Fab′-SH fragments. The SH group(s) is (are) activatedwith an excess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)bis-malemide). The RS7 MAb is convertedto Fab′-SH and then reacted with the activated CEA Fab′-SH fragment toobtain a bispecific RS7 antibody fusion protein.

A polyspecific RS7 antibody fusion protein can be obtained by adding RS7antigen binding moieties to a bispecific chimeric, humanized or humanRS7 antibody fusion protein. For example, a bispecific antibody fusionprotein can be reacted with 2-iminothiolane to introduce one or moresulfhydryl groups for use in coupling the bispecific fusion protein to athird RS7 antigen MAb or fragment, using the bis-maleimide activationprocedure described above. These techniques for producing antibodycomposites are well known to those of skill in the art. See, forexample, U.S. Pat. No. 4,925,648, which is incorporated by reference inits entirety.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody fusion proteins have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody fusion proteins containing a Fab′ portion specificto each of the original epitopes. General techniques for the preparationof antibody fusion proteins may be found, for example, in Nisonoff etal., Arch Biochem. Biophys. 93: 470 (1961), Hämmerling et al., 1 Exp.Med. 128: 1461 (1968), and U.S. Pat. No. 4,331,647. Contemplated in thepresent invention is an antibody fusion protein or fragment thereofcomprising at least one first anti-EGP-1 MAb or fragment thereof and atleast one second MAb or fragment thereof, other than the anti-EGP-1 MAbsor fragments thereof of the present invention.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,and antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody, which has at least one free aminefunction. This results in an initial Schiff base (mime) linkage, whichis preferably stabilized by reduction to a secondary amine, e.g., byborohydride reduction, to form the final composite. Such site-specificlinkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958,and for larger addends in U.S. Pat. No. 4,699,784—incorporated byreference.

ScFvs with linkers greater than 12 amino acid residues in length (forexample, 15- or 18-residue linkers) allow interacting between the V_(H)and V_(L) domains on the same chain and generally form a mixture ofmonomers, dimers (termed diabodies) and small amounts of higher massmultimers, (Kortt et al., Eur. J. Biochem. (1994) 221: 151-157). ScFvswith linkers of 5 or less amino acid residues, however, prohibitintramolecular pairing of the V_(H) and V_(L) domains on the same chain,forcing pairing with V_(H) and V_(L) domains on a different chain.Linkers between 3- and 12-residues form predominantly dimers (Atwell etal., Protein Engineering (1999) 12: 597-604). With linkers between 0 and2 residues, trimeric (termed triabodies), tetrameric (termedtetrabodies) or higher oligomeric structures of scFvs are formed;however, the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of the V-domains, in addition tothe linker length. For example, scFvs of the anti-neuraminidase antibodyNC 10 formed predominantly trimers (V_(H) to V_(L) orientation) ortetramers (V_(L) to V_(H) orientation) with 0-residue linkers (Dolezalet al., Protein Engineering (2000) 13: 565-574). For scFvs constrictedfrom NC10 with 1- and 2-residue linkers, the V_(H) to V_(L) orientationformed predominantly diabodies (Atwell et al., Protein Engineering(1999) 12: 597-604); in contrast, the V_(L), to V_(H) orientation formeda mixture of tetramers, trimers, dimers, and higher mass multimers(Dolezal et al., Protein Engineering (2000) 13: 565-574). For scFvsconstructed from the anti-CD 19 antibody HD37 in the V_(H) to V_(L),orientation, the 0-residue linker formed exclusively trimers and the1-residue linker formed exclusively tetramers (Le Gall et al., FEBSLetters (1999) 453: 164-168).

The RS7 antibodies and fragments thereof of the present invention canalso be used to produce antigen-specific diabodies, triabodies andtetrabodies, which are multivalent but monospecific. The non-covalentassociation of two or more scFv molecules can form functional diabodies,triabodies and tetrabodies. Monospecific diabodies are homodimers of thesame scFv, where each scFv comprises the V_(H) domain from the selectedantibody connected by a short linker to the V_(L) domain of the sameantibody. A diabody is a bivalent dimer formed by the non-covalentassociation of two scFvs, yielding two Fv binding sites. A triabodyresults from the formation of a trivalent trimer of three scFvs,yielding three binding sites, and a tetrabody is a tetravalent tetramerof four scFvs, resulting in four binding sites. Several monospecificdiabodies have been made using an expression vector that contains arecombinant gene construct comprising V_(H1)-linker-V_(L1). See Holligeret al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Atwell et al.,Molecular Immunology 33: 1301-1302 (1996); Holliger et al., NatureBiotechnology 15: 632-631 (1997); Helfrich et al., Int. J. Cancer 76:232-239 (1998); Kipriyanov et al., Int. J. Cancer 77: 763-772 (1998);Holiger et al., Cancer Research 59: 2909-2916 (1999)). Methods ofconstructing scFvs are disclosed in U.S. Pat. Nos. 4,946,778 (1990) and5,132,405 (1992). Methods of producing multivalent, monospecific bindingproteins based on scFv are disclosed in U.S. Pat. Nos. 5,837,242 (1998)and 5,844,094 (1998) and WO-98/44001 (1998). A preferred embodiment ofthe instant invention is a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites havingaffinity toward an EGP-1 target antigen and one or more hapten bindingsites having affinity towards hapten molecules.

Determining Antibody Binding Affinity

Comparative binding affinities of the mRS7, cRS7 and hRS7 antibodiesthus isolated may be determined by direct radioimmunoassay. RS7 can belabeled with ¹³¹I or ¹²⁵I using the chloramines-T method (see, forexample, Greenwood et al., Biochem. J., 89: 123 (1963) which isincorporated by reference). The specific activity of the iodinatedantibody is typically adjusted to about 10 μCi/μg. Unlabeled and labeledantibodies are diluted to the appropriate concentrations using reactionmedium (HSFM supplemented with 1% horse serum and 100 μg/ml gentamicin).The appropriate concentrations of both labeled and unlabeled antibodiesare added together to the reaction tubes in a total volume of 100 μl. Aculture of ME180 cells (a human cervical carcinoma cell line) is sampledand the cell concentration determined. The culture is centrifuged andthe collected cells washed once in reaction medium followed byresuspension in reaction medium to a final concentration of about 10⁷cells/ml. All procedures are carried out in the cold at 4° C. The cellsuspension, 100 μl, is added to the reaction tubes. The reaction iscarried out at 4° C. for 2 h with periodic gentle shaking of thereaction tubes to resuspend the cells. Following the reaction period, 5ml of wash buffer (PBS containing 1% BSA) is added to each tube. Thesuspension is centrifuged and the cell pellet washed a second time withanother 5 ml of wash buffer. Following centrifugation, the amount ofremaining radioactivity remaining in the cell pellet is determined in agamma counter (Minaxi, Packard Instruments, Sterling, Va.).

Expression Vectors

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.A promoter is a DNA sequence that directs the transcription of astructural gene. A structural gene is a DNA sequence that is transcribedinto messenger RNA (mRNA) which is then translated into a sequence ofamino acids characteristic of a specific polypeptide. Typically, apromoter is located in the 5′ region of a gene, proximal to thetranscriptional start site of a structural gene. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. An enhancer is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned RS7 antigen geneis a DNA fragment that has been separated from the genomic DNA of amammalian cell. Another example of an isolated DNA molecule is achemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism. Complementary DNA (cDNA) is asingle-stranded DNA molecule that is formed from an mRNA template by theenzyme reverse transcriptase. Typically, a primer complementary toportions of mRNA is employed for the initiation of reversetranscription. Those skilled in the art also use the term “cDNA” torefer to a double-stranded DNA molecule consisting of such asingle-stranded DNA molecule and its complementary DNA strand.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance. Arecombinant host may be any prokaryotic or eukaryotic cell that containseither a cloning vector or expression vector. This term also includesthose prokaryotic or eukaryotic cells that have been geneticallyengineered to contain the cloned gene(s) in the chromosome or genome ofthe host cell. The term expression refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

Humanized, Human and Chimeric RS7 Antibodies Use for Treatment andDiagnosis

Contemplated in the present invention is a method of diagnosing ortreating a malignancy in a subject comprising administering to thesubject a therapeutically effective amount of a therapeutic conjugatecomprising an EGP-1 MAb or fragment thereof or an antibody fusionprotein or fragment thereof, wherein the EGP-1MAb or fragment thereof orantibody fusion protein or fragment thereof is bound to at least onetherapeutic agent and then formulated in a pharmaceutically suitableexcipient. It is also contemplated that an unconjugated (naked) EGP-1MAb or fusion construct with other antigen-binding moieties also can besued as a therapeutic for cancer cells expressing EGP-1. Theseunconjugated' antibodies may be given advantageously in combination withother therapeutic modalities, such as chemotherapy, radiotherapy, and/orimmunotherapy, either together or in various sequences and schedules.Also preferred is a method for diagnosing or treating cancer,comprising: administering a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites toward aEGP-1 antigen and one or more hapten binding sites to a subject in needthereof, waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and thenadministering to the subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to thebinding site of the multivalent, multispecific antibody or fragmentthereof. In a preferred embodiment, the cancer is a lung, breast, headand neck, ovarian, prostate, bladder or colon cancer.

Hybridoma technology for the production of monoclonal antibodies (MAbs)has provided a method for the production of molecular probes capable oflocating or killing cancer cells. Tumor imaging techniques usingradiolabeled MAbs have been used to delineate cancerous invasion in anumber of malignancies. In experimental animals and in humans,antibodies have been used for the radioimmunodetection ofcarcinoembryonic antigen in diverse tumors that express carcinoembryonicantigen, and also tumors such as melanoma, colon carcinoma, and breastcarcinoma with other targeting antibodies. Goldenberg et al., CancerRes. 40: 2984 (1980); Hwang et al., Cancer Res. 45: 4150 (1985);Zalcberg et al., J. Nat'l Cancer Inst. 71: 801 (1983); Colcher et al.,Cancer Res. 43: 736 (1983); (Larson et al., J. Nucl. Med. 24: 123(1983); DeLand et al., Cancer Res. 40: 3046 (1980); Epenetos et al.,Lancet 2: 999 (1982).

The use of MAbs for in vitro diagnosis is well known. See, for example,Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example, MAbs canbe used to detect the presence of a tumor-associated antigen in tissuefrom biopsy samples. MAbs also can be used to measure the amount oftumor-associated antigen in clinical fluid samples using techniques suchas radioimmunoassay, enzyme-linked immunosorbent assay, and fluorescenceimmunoassay.

Conjugates of tumor-targeted MAbs and toxins can be used to selectivelykill cancer cells in vivo (Spalding, Bio/Technology 9(8): 701 (1991);Goldenberg, Scientific American Science & Medicine 1(1): 64 (1994)). Forexample, therapeutic studies in experimental animal models havedemonstrated the anti-tumor activity of antibodies carrying cytotoxicradionuclides. (Goldenberg et al., Cancer Res. 41: 4354 (1981), Cheunget al., J. Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch et al., JNucl. Med. 30: 531 (1989)). Also, see Stein et al., Antibody Immunoconj.Radiopharm. 4: 703 (1991), which is fully incorporated by reference.Moreover, Phase-I therapeutic trials with some of these MAbs have beeninitiated for treatment of lymphoma, melanoma, and other malignancies.See, for example, DeNardo et al., Int. J. Cancer Suppl. 3: 96 (1988),and Goldenberg et al., J. Clin. Oncol. 9: 548 (1991).

Humanized, chimeric and fully human antibodies and fragments thereof aresuitable for use in therapeutic methods and diagnostic methods.Accordingly, contemplated in the present invention is a method ofdelivering a diagnostic or therapeutic agent, or a combination thereof,to a target comprising (i) providing a composition that comprises ananti-EGP-1 antibody and (ii) administering to a subject in need thereofthe diagnostic or therapeutic antibody conjugate. Preferably, thechimeric, humanized and fully human RS7 antibodies and fragments thereofof the present invention are used in methods for treating malignancies.

Also described herein is a cancer cell targeting diagnostic ortherapeutic conjugate comprising an antibody component comprising ananti-EGP-1 mAb or fragment thereof or an antibody fusion protein orfragment thereof that hinds to the cancer cell, wherein the antibodycomponent is bound to at least one diagnostic or at least onetherapeutic agent. Preferably, the diagnostic conjugate comprises atleast a photoactive diagnostic agent or an MRI contrast agent. Stillpreferred, the diagnostic agent is a radioactive label with an energybetween 60 and 4,000 keV.

The compositions for treatment contain at least one naked or conjugatedhumanized, chimeric or human RS7 antibody alone, or in combination withother naked or conjugated humanized, chimeric, human or other antibodiesof the present invention, or other naked or conjugated humanized,chimeric or human antibodies not disclosed herein. The present inventionalso contemplates administration of a conjugated or naked antibody witha therapeutic agent such as an immunomodulator, or diagnostic agent thatis not conjugated to the anti-EGP-1 antibody. Naked or conjugatedantibodies to the same or different epitope or antigen may be alsocombined with one or more of the antibodies of the present invention.

Accordingly, the present invention contemplates the administrationanti-EGP-1 antibodies and fragments thereof alone, as a naked antibodyor antibody fragment, or administered as a multimodal therapy.Preferably, the antibody is a humanized, chimeric or fully human RS7antibody or fragment thereof. Multimodal therapies of the presentinvention further include immunotherapy with a naked anti-EGP-1 antibodysupplemented with administration of other antibodies in the form ofnaked antibodies, fusion proteins, or as immunoconjugates. For example,a humanized, chimeric or fully human RS7 antibody may be combined withanother naked humanized, chimeric RS7 or other antibody, or a humanized,chimeric RS7 or other antibody conjugated to an isotope, one or morechemotherapeutic agents, cytokines, toxins or a combination thereof. Forexample, the present invention contemplates treatment of a naked orconjugated EGP-1 or RS7 antibody or fragments thereof before, incombination with, or after other solid tumor/carcinoma associatedantibodies such as anti-EGP-2, CEA, CSAp, MUC1-4, EGFR, HER2/neu, PSA,CC49 (anti-Tag 72 antibody) and PSMA antibodies. These solid tumorantibodies may be naked or conjugated to, inter alfa, drugs, enzymes,hormones, toxins, isotopes, or immunomodulators. A fusion protein of ahumanized, chimeric or fully human RS7 antibody and a toxin or may alsobe used in this invention. Many different antibody combinations may beconstructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator. Alternatively,different naked antibody combinations may be employed for administrationin combination with other therapeutic agents, such as a cytotoxic drugor with radiation. Combinations of such antibodies can also be made,advantageously, with antisense oligonucleotides, as are known in theart. As such, the therapeutic conjugates may comprise anoligonucleotide, especially an antisense oligonucleotide that preferablyare directed against oncogenes and oncogene products of B-cellmalignancies. For example, antisense molecules inhibiting bcl-2expression that are described in U.S. Pat. No. 5,734,033 (Reed) which isincorporated by reference in its entirety, may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with a humanized RS7 antibody of the present invention.

The monospecific binding proteins described herein that are linked todiagnostic or therapeutic agents directly target RS7 positive tumors.The monospecific molecules bind selectively to targeted antigens and asthe number of binding sites on the molecule increases, the affinity forthe target cell increases and a longer residence time is observed at thedesired location. Moreover, non-antigen bound molecules are cleared fromthe body quickly and exposure of normal tissues is minimized. A use ofmultispecific binding proteins is pre-targeting RS7 positive tumors forsubsequent specific delivery of diagnostic or therapeutic agents. Theagents arc carried by histamine succinyl glycyl (HSG) containingpeptides. The murine monoclonal antibody designated 679 (an IgG1, K)binds with high affinity to molecules containing the tri-peptide moiety,HSG (Morel et al., Molecular immunology, 27, 995-1000, 1990). 679 MAbcan form a bispecific binding protein with hRS7 that binds with HSG andthe target antigen. Alternative haptens may also be utilized. Thesebinding proteins bind selectively to targeted antigens allowing forincreased affinity and a longer residence time at the desired location.Moreover, non-antigen bound diabodies are cleared from the body quicklyand exposure of normal tissues is minimized.

RS7 antibodies and fragments thereof can be used to treat mammaliandisorders such as cancer. The cancer includes, but is not limited to,lung, breast, bladder, ovarian prostate and colon cancers.

Delivering a diagnostic or a therapeutic agent to a target for diagnosisor treatment in accordance with the invention includes providing theanti-EGP-1 antibody or fragments thereof with a diagnostic ortherapeutic agent and administering to a subject in need thereof withthe binding protein. Diagnosis further requires the step of detectingthe bound proteins with known techniques.

Administration of the antibodies and their fragments of the presentinvention with diagnostic or therapeutic agents can be effected in amammal by intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, perfusion through a regionalcatheter, or direct intralesional injection. When administering thebinding protein by injection, the administration may be by continuousinfusion or by single or multiple boluses. Doses in the range of 20 to800 mg/m² are feasible, with doses between 100 and 500 mg/m² preferably,for therapy, and commensurately lower doses recommended for diagnosticimaging, such as 0.5 mg to 100 mg/patient. Such doses may be repeated atdifferent frequencies, depending on the clinical situation and patienttolerance.

The antibody with the diagnostic or therapeutic agent may be provided asa kit for human or mammalian therapeutic and diagnostic use in apharmaceutically acceptable injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.The preparation preferably will be sterile, especially if it is intendedfor use in humans. Optional components of such kits include stabilizers,buffers, labeling reagents, radioisotopes, paramagnetic compounds,second antibody for enhanced clearance, and conventional syringes,columns, vials and the like.

Naked Antibody Therapy

A therapeutically effective amount of the naked chimeric, humanized andfully human RS7 antibodies, or their fragments, can be formulated in apharmaceutically acceptable excipient. The efficacy of the nakedchimeric, humanized and fully human RS7 antibodies can also be enhancedby supplementing these naked antibodies with one or more other nakedantibodies, with one or more immunoconjugates of chimeric, humanized andfully human RS7 antibodies conjugated to a therapeutic agent, such as adrug, toxin, immunomodulator, hormone, growth factor, enzyme ortherapeutic radionuclides, or with one or more therapeutic agent,including a drug, toxin, immunomodulator, hormone, growth factor,enzyme, oligonucleotide, or therapeutic radionuclide, administeredconcurrently or sequentially or according to a prescribed dosingregimen, with the RS7 antibodies or fragments thereof.

In a preferred embodiment, the naked or conjugated RS7 antibodies of thepresent invention are combined with at least one cancer drug. Suchcombination therapy can improve the effect of the drug or lower drugdose that is needed. For example, the IC₅₀ value was determined forDox-RS7 and 2P-Dox-RS7 on a lung cancer cell line, Calu3, and two breastcancer cell lines, MDA468 and T47D, respectively. Calu3 and T47D cellsare positive for an EGP-1 antigen and negative for a CEA antigen, andMDA468 is positive for both the EGP-1 and CEA antigens. Results indicatethat the IC₅₀ value for Dox-RS7 is 0.04 μg/ml and for 2P-Dox-RS7 is0.023 μg/ml. Therefore, conjugating a naked, human, humanized orchimeric anti-EGP-1 antibody or fragment of the present invention to aparticular drug, such as 2P-Dox may help overcome multidrug resistance.This is also possible when the antibody is given in a combination with aparticular drug, as described.

RS7 Immunoconjugates

The present invention also contemplates the use of humanized, chimericand human RS7 antibodies and fragments thereof for therapy. Theobjective of immunotherapy is to deliver cytotoxic doses ofradioactivity, toxin, cytokine, enzyme, or hormone, or drug to targetcells, while minimizing exposure to non-target tissues. The RS7 antigenbinding proteins of the present invention can be used to treat a varietyof tumors, such as of the lung, breast, bladder, ovary, uterus, stomach,and prostate.

Any of the antibodies or antibody fusion proteins and fragments thereofof the present invention can be conjugated with one or more therapeuticor diagnostic agents. Generally, one therapeutic or diagnostic agent isattached to each antibody or antibody fragment but more than onetherapeutic agent or diagnostic agent can be attached to the sameantibody or antibody fragment. If the Fc region is absent (for examplewhen the antibody used as the antibody component of the immunoconjugateis an antibody fragment), it is possible to introduce a carbohydratemoiety into the light chain variable region of a full-length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al.,U.S. Pat. No. 6,254,868, all of which are incorporated in their entiretyby reference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313,all of which are incorporated in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate. Also, a chelator such as DTPA (such as Mx-DTPA), DOTA, TETA,or NOTA can be attached to the antibody.

The antibody fusion proteins of the present invention comprise two ormore antibodies or fragments thereof and each of the antibodies orfragments that compose this fission protein can contain a therapeuticagent or diagnostic agent. Additionally, one or more of the antibodiesor fragments of the antibody fusion protein can have more than onetherapeutic of diagnostic agent attached. Further, the therapeuticagents do not need to be the same but can be different therapeuticagents, for example, one can attach a drug and a radioisotope to thesame fusion protein. Particularly, an IgG can be radiolabeled with ¹³¹Iand attached to a drug. The ¹³¹I can be incorporated into the tyrosineof the IgG and the drug attached to the epsilon amino group of the IgGlysines. Both therapeutic and diagnostic agents also can be attached toreduced SH groups and to the carbohydrate side chains.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Thetherapeutic agents recited here are those agents that also are usefulfor administration separately with the naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes,antimetabolites, alkylating agents, antibiotics, substituted urea,enzymes, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoicagents, particularly doxorubicin, doxorubicin analogs, methotrexate,taxol, CPT-11, camptothecans, and others from these and other classes ofanticancer agents, methyl hydrazine derivative, adrenocorticalsuppressant, antagonist, endostatin, taxol, and the like. Other usefulcancer chemotherapeutic drugs for the preparation of immunoconjugatesand antibody fusion proteins include nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, tyrosine kinase inhibitors, such asthose that inhibit a EGF-receptor tyrosine kinase, a BCR ABL tyrosinekinase or a VEGF-receptor tyrosine kinase, and the like. Suitablechemotherapeutic agents are described in Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and in Goodman andGilman's The Pharmacological Basis of Therapeutics, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an immunoconjugate of the RS7 and hRS7antibodies of the present invention. Other toxins suitably employed inthe preparation of such conjugates or other fusion proteins, includericin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastanet al., Cell 47:641 (1986), and Goldenberg, CA—A Cancer Journalfor Clinicians 44:43 (1994). Additional toxins suitable for use in thepresent invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of the EGP-1, RS7 and hRS7immunoconjugate, or be administered unconjugated to the chimeric,humanized or human RS7 antibodies or fragments thereof of the presentinvention. As used herein, the teen “immunomodulator” includescytokines, stem cell growth factors, lymphotoxins, such as tumornecrosis factor (TNF), and hematopoietic factors, such as interleukins(e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, andIL-21), colony stimulating factors (e.g., granulocyte-colony stimulatingfactor (G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF)), interferons (e.g., interferons-α, -β and -γ), the stem cellgrowth factor designated “S1 factor,” erythropoietin and thrombopoietin,or a combination thereof. Examples of suitable immunomodulator moietiesinclude IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-α, andthe like. Alternatively, subjects can receive naked EGP-1 or RS7antibodies and a separately administered cytokine, which can beadministered before, concurrently or after administration of the nakedRS7 antibodies. The RS7 antibody may also be conjugated to theimmunomodulator. The immunomodulator may also be conjugated to a hybridantibody consisting of one or more antibodies binding to differentantigens.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well known in theart. See, for example, Wong, Chemistry of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Furthermore, a radiolabeled antibody, immunoconjugate, or fragmentsthereof may comprise a γ-emitting radioisotope or a positron-emitteruseful for diagnostic imaging. Suitable radioisotopes, particularly inthe energy range of 25 to 4,000 keV, include ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y,⁶²Cu, ⁶⁴Cu ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, andthe like. See for example, U.S. patent application entitled “LabelingTargeting Agents with Gallium-68”-Inventors G. L. Griffiths and W. J.McBride, (U.S. Provisional Application No. 60/342,104 (expired), nowU.S. Pat. No. 7,011,816), which discloses positron emitters, such as¹⁸F, ⁶⁸Ga, ^(94m)Tc and the like, for imaging purposes and which isincorporated in its entirety by reference. Preferably, the energy rangefor diagnostic and therapeutic radionuclides is 25-4,000_keV. Otheruseful radionuclides include ⁹⁰Y, ¹¹¹In, ¹²⁵I, ³H, ³⁵S, ¹⁴C, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ²¹¹At, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br,^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(94m)Te, ^(121m)Te,^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹¹¹Ag, ¹⁹⁷Pt, ¹⁰⁹Pd, ³²P,³³P, ⁴⁷Sc, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ¹⁸F, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ⁸⁶Y, ¹⁶⁹Yb, ¹⁶⁶Dy,²¹²Pb, and ²²³Ra.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5 hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to an RS7antigen binding protein using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an RS7 antigen binding protein usingdiethylenetriaminepentaacetic acid (DTPA). Moreover, a method for thedirect radiolabeling of the RS7 MAb with ¹³¹I is described by Stein etal. (1991), supra, and the patent by Govindan et al., WO 9911294A1entitled “Stable Radioiodine Conjugates and Methods for TheirSynthesis,” and is incorporated herein by reference in their entirety.

The RS7 antibodies or fragments thereof of the present invention thathave a boron addend-loaded carrier for thermal neutron activationtherapy will normally be effected in similar ways. However, it will beadvantageous to wait until non-targeted RS7 immunoconjugate clearsbefore neutron irradiation is performed. Clearance can be acceleratedusing an antibody that binds to the RS7 antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to RS7 antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the RS7antibody. After administration of the RS7 antibody conjugate, a boronaddend is activated by thermal neutron irradiation and converted toradioactive atoms that decay by α-emission to produce highly toxic,short-range effects.

Furthermore, the present invention includes methods of diagnosing cancerin a subject. Diagnosis may be accomplished by administering adiagnostically effective amount of a diagnostic conjugate, formulated ina pharmaceutically suitable excipient, and detecting said label. Forexample, radioactive and non-radioactive agents can be used asdiagnostic agents. A suitable non-radioactive diagnostic agent is acontrast agent suitable for magnetic resonance imaging, computedtomography or ultrasound. Magnetic imaging agents include, for example,non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, when used along with theantibodies of the invention. See U.S. Ser. No. 09/921,290 filed on Oct.10, 2001 (PGP No. US 2002/0041847 A1), which is incorporated in itsentirety by reference.

Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising (i) performing an in vitro diagnosis assay on aspecimen from the subject with a composition comprising a nakedanti-EGP-1 MAb or fragment thereof or a naked antibody fusion protein orfragment thereof. For example, RT-PCR and immunoassay in vitro diagnosismethods can be used to detect the presence of minute amounts of EGP-1 intissues, blood and other body fluids as a useful diagnostic/detectionmethod. Immunohistochemistry can be used to detect the presence of EGP-1in a cell or tissue. Preferably, the malignancy that is being diagnosedis a cancer. Most preferably, the cancer is selected from the group oflung, prostate, ovarian, breast, colon and bladder.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibody fusionprotein. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Joni et al. (eds.), PhotodynamicTherapy of Tumors and Other Diseases (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mewet al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem.,Photochem. Photobiol. 46:83 (1987); Hasanet al., Prog. Clin. Biol. Res.288:471 (1989); Tatsutaet al., Lasers Surg. Med. 9:422 (1989);Pelegrinet al., Cancer 67:2529 (1991). However, these earlier studiesdid not include use of endoscopic therapy applications, especially withthe use of antibody fragments or subfragments. Thus, the presentinvention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

Contrast agents such as a MRI contrast agent, a paramagnetic ion and anultrasound enhancing agent are also contemplated in the presentinvention. For example, gadolinium ions, lanthanum ions, manganese ionsor other comparable label, CT contrast agents, and ultrasound contrastagents are suitable for use in the present invention. In a preferredembodiment, the ultrasound enhancing agent is a liposome that comprisesa humanized RS7 IgG or fragment thereof. Also preferred, the liposome isgas filled.

For purposes of therapy, the RS7 antibodies and fragments thereof of thepresent invention are administered to a patient in a therapeuticallyeffective amount. An antibody is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

In Vitro Diagnosis

The present invention contemplates the use of RS7 antibodies, includingRS7 and hRS7 antibodies and fragments thereof, to screen biologicalsamples in vitro for the presence of the RS7 antigen. In suchimmunoassays, the RS7 antibody may be utilized in liquid phase or boundto a solid-phase carrier, as described below. Also, see Stein et al.(1993), supra, and Stein et al., Cancer Res. 49: 32 (1989), which isfully incorporated by reference.

One example of a screening method for determining whether a biologicalsample contains the RS7 antigen is the radioimmunoassay (RIA). Forexample, in one form of RIA, the substance under test is mixed with RS7antigen MAb in the presence of radiolabeled RS7 antigen. In this method,the concentration of the test substance will be inversely proportionalto the amount of labeled RS7 antigen bound to the MAb and directlyrelated to the amount of free labeled RS7 antigen. Other suitablescreening methods will be readily apparent to those of skill in the art.

Alternatively, in vitro assays can be performed in which an RS7 antigenbinding protein is bound to a solid-phase carrier. For example, MAbs canbe attached to a polymer, such as aminodextran, in order to link the MAbto an insoluble support such as a polymer-coated bead, a plate or atube.

Other suitable in vitro assays will be readily apparent to those ofskill in the art. The specific concentrations of detectably labeled RS7antigen binding protein and RS7 antigen, the temperature and time ofincubation, as well as other assay conditions maybe varied, depending onvarious factors including the concentration of the RS7 antigen in thesample, the nature of the sample, and the like. The binding activity ofa sample of RS7 antigen binding protein may be determined according towell known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

The presence of the RS7 antigen in a biological sample can be determinedusing an enzyme-linked immunosorbent assay (ELISA). In the directcompetitive ELISA, a pure or semipure antigen preparation is bound to asolid support that is insoluble in the fluid or cellular extract beingtested and a quantity of detectably labeled soluble antibody is added topermit detection and/or quantitation of the binary complex formedbetween solid-phase antigen and labeled antibody.

In contrast, a “double-determinant” ELISA, also known as a “two-siteELISA” or “sandwich assay,” requires small amounts of antigen and theassay does not require extensive purification of the antigen. Thus, thedouble-determinant ELISA is preferred to the direct competitive ELISAfor the detection of an antigen in a clinical sample. See, for example,the use of the double-determinant ELISA for quantitation of the c-myconcoprotein in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a double-determinant ELISA, a quantity of unlabeled MAb or antibodyfragment (the “capture antibody”) is bound to a solid support, the testsample is brought into contact with the capture antibody, and a quantityof detectably labeled soluble antibody (or antibody fragment) is addedto permit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. An antibodyfragment is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,and the like. In the present context, an antibody fragment is a portionof an RS7 MAb that binds to an epitope of the RS7 antigen. The term“antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker. Anantibody fusion protein is a polyspecific antibody compositioncomprising at least two substantially monospecific antibodies orantibody fragments, wherein at least two of the antibodies or antibodyfragments bind to different epitopes of the RS7 antigen. An RS7 fusionprotein also includes a conjugate of an antibody fusion protein with adiagnostic or therapeutic agent. The term RS7 antibody includeshumanized, chimeric, human and murine antibodies, antibody fragmentsthereof, immunoconjugates and fragments thereof and antibody fusionproteins and fragments thereof.

Methods of performing a double-determinant ELISA are well-known. See,for example, Field et al., supra, Spandidos et al., supra, and Moore etal., “Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAKSystem,” in Methods in Molecular Biology, Vol. 10, pages 273-281 (TheHumana Press, Inc. 1992). For example, in one method for the detectionof RS7 antigen using the double-determinant ELISA, finely minced tissuefrom a biopsy sample is lyophilized and resuspended in lysis buffer (100mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 1% nonidet-p40 (NP40), 0.6μl/ml aprotinin, 0.2 mM phenyl methyl sulphonyl fluoride, 0.1 μg/mlleupeptin and 1 mM EDTA at a concentration of 10-20 mg tissue (wetweight) per 500 μl solution. The suspension is incubated for 60 minuteson ice, and then sonicated for approximately six 10-second intervals.Insoluble material is removed by centrifugation.

The soluble extract is added to microliter plate wells containing anadsorbed RS7 antigen MAb as the capture antibody. Captured RS7 antigenis then recognized by a second RS7 antigen MAb, which has been coupledwith alkaline phosphatase. The amount of bound alkaline phosphatase,proportional to the amount of RS7 antigen in the extract, is detectedcolormetrically using a chromogenic substrate, such asp-nitrophenylphosphate.

Alternatively, a double-determinant ELISA for the RS7 antigen can beperformed using horse radish peroxidase. Other variations of samplepreparation and the double-determinant ELISA can be devised by those ofskill in the art with routine experimentation.

In the double-determinant ELISA, the soluble antibody or antibodyfragment must bind to an RS7 epitope that is distinct from the epitoperecognized by the capture antibody. For example, the soluble antibodycan be the RS7 MAb, while the capture antibody can be MR23.Alternatively, the soluble antibody can be MR23, while the captureantibody can be the RS7 MAb.

The double-determinant ELISA can be performed to ascertain whether theRS7 antigen is present in a biopsy sample. Alternatively, the assay canbe performed to quantitate the amount of RS7 antigen that is present ina clinical sample of body fluid. The quantitative assay can be performedby including dilutions of purified RS7 antigen. A method for purifyingthe RS7 antigen is illustrated below.

The RS7 MAbs and fragments thereof of the present invention also aresuited for the preparation of an assay kit. Such a kit may comprise acarrier means that is compartmentalized to receive in close confinementone or more container means such as vials, tubes and the like, each ofsaid container means comprising the separate elements of theimmunoassay.

For example, there may be a container means containing the captureantibody immobilized on a solid phase support, and a further containermeans containing detectably labeled antibodies in solution. Furthercontainer means may contain standard solutions comprising serialdilutions of RS7 antigen. The standard solutions of RS7 antigen may beused to prepare a standard curve with the concentration of RS7 antigenplotted on the abscissa and the detection signal on the ordinate. Theresults obtained from a sample containing RS7 antigen may beinterpolated from such a plot to give the concentration of RS7 antigenin the biological sample.

RS7 antibodies and their fragments of the present invention also can beused to detect the presence of the RS7 antigen in tissue sectionsprepared from a histological specimen. Such in situ detection can beused to determine the presence of the RS7 antigen and to determine thedistribution of the RS7 antigen in the examined tissue. In situdetection can be accomplished by applying a detectably-labeled RS7antigen binding protein to frozen tissue sections. Studies indicate thatthe RS7 antigen is not preserved in paraffin-embedded sections. Stein etal. (1993), supra. General techniques of in situ detection are wellknown to those of ordinary skill. See, for example, Ponder, “CellMarking Techniques and Their Application,” in Mammalian Development: APractical Approach, 113-38 Monk (ed.) (IRL Press 1987), and Coligan atpages 5.8.1-5.8.8. Also, see Stein et al. (1989), supra, and Stein etal. (1993), supra.

RS7 antibodies and their fragments can be detectably labeled with anyappropriate detection agent, for example, a radioisotope, an enzyme, afluorescent label, a chemiluminescent label, a bioluminescent label or aparamagnetic label. Methods of making and detecting suchdetectably-labeled RS7 antigen binding proteins are well-known to thoseof ordinary skill in the art, and are described in more detail below.

The marker moiety can be a radioisotope that is detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to molecule that will generate a signal underpredetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels. As used herein, adiagnostic or therapeutic agent is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate, which is usefulfor diagnosis and for therapy. Examples of diagnostic or therapeuticagents include drugs, toxins, chelators, dyes, chromagens, boroncompounds, and marker moieties. Isotopes that are particularly usefulfor the purpose of the present invention are ³H, ¹³¹I, ³⁵S, ¹⁴C, andpreferably ¹²⁵I. Examples of other radionuclides are, for example, ⁹⁰Y,¹¹¹In, ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, and ²¹¹At.Additional radionuclides are also available as diagnostic andtherapeutic agents. Suitable diagnostic imaging isotopes are usually inthe range of 25 to 4,000 keV, while suitable therapeutic radionuclidesare usually in the range of 60 to 700 keV.

The RS7 antibodies and their fragments of the present invention also canbe labeled with a fluorescent compound. The presence of afluorescently-labeled MAb is determined by exposing the RS7 antigenbinding protein to light of the proper wavelength and detecting theresultant fluorescence. Fluorescent labeling compounds includefluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Fluorescently-labeledRS7 antigen binding proteins are particularly useful for flow cytometryanalysis.

Alternatively, RS7 antibodies and their fragments can be detectablylabeled by coupling the RS7 antigen binding protein to achemiluminescent compound. The presence of the chemiluminescent-taggedMAb is determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of chemiluminescentlabeling compounds include luminol, isoluminol, an aromatic acridiniumester, an imidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label RS7 antibodiesand fragments thereof the present invention. Bioluminescence is a typeof chemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, RS7 antibodies and fragments thereof can be detectablylabeled by linking the RS7 antibody to an enzyme. When the RS7antibody-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label RS7 antibody include malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

RS7 antibodies, fusion proteins, and fragments thereof also can belabeled with paramagnetic ions for purposes of in vivo diagnosis.Contrast agents that are particularly useful for magnetic resonanceimaging comprise Gd, Mn, Dy or Fe ions. RS7 antibodies and fragmentsthereof can also be conjugated to ultrasound contrast/enhancing agents.For example, the ultrasound contrast agent is a liposome that comprisesa humanized RS7 IgG or fragment thereof. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

In a related vein, a bispecific antibody can be conjugated to a contrastagent. For example, the bispecific antibody may comprise more than oneimage-enhancing agent for use in ultrasound imaging. In a preferredembodiment, the contrast agent is a liposome. Preferably, the liposomecomprises a bivalent DTPA-peptide covalently attached to the outsidesurface of the liposome. Still preferred, the liposome is gas filled.

Those of skill in the art will know of other suitable labels that can beemployed in accordance with the present invention. The binding of markermoieties to RS7 antibodies can be accomplished using standard techniquesknown to the art. Typical methodology in this regard is described byKennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim.Acta 81: 1 (1977), Shih et al., Int'l J. Cancer 46: 1101 (1990), Steinet al. (1990), supra, and Stein et al. (1993), supra. Also, seegenerally, Coligan.

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the RS7antigen including tumors of the lung, breast, bladder, ovary, uterus,stomach, and prostate.

In Vivo Diagnosis

The present invention also contemplates the use of RS7 antibodies for invivo diagnosis. The method of diagnostic imaging with radiolabeled MAbsis well-known. In the technique of immunoscintigraphy, for example,antibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), Radiolabeled Monoclonal Antibodies for Imaging andTherapy (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in Remington's Pharmaceutical Sciences, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in Biotechnology andPharmacy, 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

For diagnostic imaging, radioisotopes may be bound to the RS7 antibodyeither directly, or indirectly by using an intermediary functionalgroup. Useful intermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see Shih et al., supra, and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope, which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to RS7 antibodyand are appropriate for diagnostic imaging include ^(99m)Tc and ¹¹¹In.

Pharmaceutically Suitable Excipient

Additional pharmaceutical methods may be employed to control theduration of action of an RS7 antibody in a therapeutic application.Control release preparations can be prepared through the use of polymersto complex or adsorb the RS7 antibody. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release of an RS7 antibody from such a matrix depends upon themolecular weight of the RS7 antibody, the amount of RS7 antibody withinthe matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Remington's Pharmaceutical Sciences, 18th ed.(1990).

The humanized, chimeric and human RS7 antibodies to be delivered to asubject can consist of the antibody alone, immunoconjugate, fusionprotein, or can comprise one or more pharmaceutically suitableexcipients, one or more additional ingredients, or some combination ofthese.

The immunoconjugate, naked antibody, fusion protein, and fragmentsthereof of the present invention can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby theimmunoconjugate or naked antibody is combined in a mixture with apharmaceutically suitable excipient_Sterile phosphate-buffered saline isone example of a pharmaceutically suitable excipient. Other suitableexcipients are well known to those in the art. See, for example, Anselet al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), Remington'sPharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion protein, naked antibody, andfragments thereof may also be administered to a mammal subcutaneously oreven by other parenteral routes. In a preferred embodiment, theanti-EGP-1 antibody or fragment thereof is administered in a dosage of10 to 2000 milligrams protein per dose, and preferably is repeatedlyadministered. Moreover, the administration may be by continuous infusionor by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

The RS7 antibodies of the present invention can be formulated accordingto known methods to prepare pharmaceutically useful compositions,whereby RS7 antibodies are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers are wellknown to those in the art. See, for example, Remington's PharmaceuticalSciences, 18th Ed. (1990).

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject for the present invention is usually a human,although a non-human animal subject is also contemplated. An antibodypreparation is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compositions andprocesses of this invention. Thus, it is intended that the presentinvention cover such modifications and variations, provided they comewithin the scope of the appended claims and their equivalents.

The disclosure of all publications, patents and patent applicationscited above are expressly incorporated herein by reference in theirentireties to the same extent as if each were incorporated by referenceindividually.

The examples below are illustrative of embodiments of the currentinvention and should not be used, in any way, to limit the scope of theclaims.

EXAMPLE 1 Construction of a Chimeric RS7 Antibody

Molecular Cloning of RS7 Vκ and VH Genes

Total cytoplasmic RNA and m NA was prepared from RS7-producing hybridomacells. The genes encoding Vκ and VH sequences were cloned by RT-PCR and5′RACE and the sequences were determined by DNA sequencing. Multipleindependent clones were sequenced to eliminate possible errors resultingfrom the PCR reaction. Sequence analyses revealed presence of two Vκ (#1and #23) and one VH (RS7VH) transcripts. Combining each of the putativemurine Vκ with the VH, two chimeric Abs (cAbs), containing humanconstant region domains were generated and expressed in Sp2/0 cells bytransfection. cAb-producing clones were identified by screening the cellculture supernatants of the transfected cell clones by ELISA. Positiveclones were expanded and cAbs were purified from the cell culturesupernatants. The Ag-binding assay showed that only the cAb composed ofVκ#23 and VH, cAb-Vκ#23, bound to microwells coated with the crudemembrane fraction of ME180, a human cervical carcinoma cell (ATCC,Rockville, Md.) (FIG. 1). The cAb with the combination of Vκ#1 and VH,cAb-Vκ#1, did not show binding to the Ag-coated wells. Therefore, theimmunoreactive cAb (with Vκ#23) was designated as cRS7. The clonedmurine V_(H) and the functional Vκ (#23) sequences as the final PCRproducts were designated as RS7Vκ (FIG. 2A, SEQ ID NO: 1) and RS7VH(FIG. 2B, SEQ ID NO: 3), respectively.

Binding Activity Assay for RS7 Abs

A competitive ELISA binding assay was used to evaluate the bindingaffinity of engineered cRS7. Briefly, constant amount of biotinylatedmurine RS7 is mixed with varying concentrations (0.01-100 μg/ml) oftesting Abs (RS7 or cRS7), and added into Ag-coated microwells, andincubated at room temperature for 1 h. After washing, HRP conjugatedstreptavidin is added and incubated for 1 h at room temperature. Theamount of HRP-conjugated streptavidin bound to the Ag-bound biotinylatedRS7 was revealed by reading OD₄₉₀ after the addition of a substratesolution containing 4 mM ortho-phenylenediamine dihydrochloride and0.04% H₂O₂. By this type of competitive Ag-binding assay, it wasrevealed that cRS7 and murine RS7 competed equally well for the bindingof biotinylated murine RS7 to the antigen coated wells, thus confirmedthe authenticity of the Vκ and VH sequences obtained (FIG. 1).

EXAMPLE 2 Method of hRS7 Antibody Construction

Sequence Design of hRS7 V Genes

By searching the human Vκ and VH sequences in the Kabat database, theFRs of RS7 Vκ (SEQ ID NO: 2) and VH (SEQ ID NO: 4) were found to exhibitthe highest degree of sequence homology to human SA-1A'cl Vκ (SEQ ID NO:5) and RF-TS3 VH (SEQ ID NO: 8), respectively. One exception is the FR4of RS7VH, which showed the highest sequence homology with that of NEWMVH (SEQ ID NO: 6. Therefore human SA-1A'CL framework sequences were usedas the scaffold for grafting the CDRs of RS7Vκ (FIG. 3A, SEQ ID NO: 5;SEQ ID NO: 2; SEQ ID NO: 7), and a combination of RF-TS3 and NEWMframework sequences were used for RS7V_(H) (FIG. 4, SEQ ID NO: 11; SEQID NO: 12). There are a number of amino acid changes in each chainoutside of the CDR regions when compared to the starting human antibodyframeworks. Several amino acid residues in murine FRs that flank theputative CDRs were maintained in the reshaped hRS7 Fv based on theguideline previously established Qu, Z., Losman, M. J., Eliassen, K. C.,Hansen, H. J., Goldenberg, D. M., and Leung, S. O. (1999). Humanizationof Immu31, an alphafetoprotein-specific antibody. Clin. Cancer Res. 5,3095s-3100s. These residues are S20, D60, V85, and A100 of RS7Vκ andK38, K46, A78, and F91 of RS7VH (FIG. 3A, SEQ ID NO: 5; SEQ ID NO: 2;SEQ ID NO: 7) and 3B, SEQ ID NO: 8; SEQ ID NO: 4; SEQ ID NO: 10).

Construction of hRS7 V Sequences

A modified strategy as described by Leung et al. (Leung, S. O., Shevitz,J., Pellegrini, M. C., Dion, A. S., Shih, L. B., Goldenberg, D. M., andHansen, H. J. (1994) Chimerization of LL2, a rapidly internalizingantibody specific for B cell lymphoma. Hybridoma, 13: 469-476) was usedto construct the designed VL and VH genes for hRS7 using a combinationof long oligonucleotide syntheses and PCR as illustrated in FIG. 4 (SEQID NO: 11-12; SEQ ID NO: 13-14). For the construction of the hRS7 VHdomain, two long oligonucleotides, hRS7VHA (176-mer) and hRS7VHB(168-mer) were synthesized on an automated DNA synthesizer (AppliedBiosystem).

hRS7VHA (SEQ ID NO: 19) represents nt 23 to 198 of the hRS7VH domain

(SEQ ID NO: 19) 5′-GGTCTGAGTT GAAGAAGCCT GGGGCCTCAG TGAAGGTTTCCTGCAAGGCT TCTGGATACA CCTTCACAAA CTATGGAATG AACTGGGTGA AGCAGGCCCCTGGACAAGGG CTTAAATGGA TGGGCTGGAT AAACACCTAC ACTGGAGAGC CAACATATACTGATGACTTC AAGGGA-3′hRS7VHB (SEQ ID NO: 20) represents the minus strand of the hRS7VH domaincomplementary to nt 174 to 340.

(SEQ ID NO: 20) 5′-ACCCTTGGCC CCAGACATCG AAGTACCAGT AGCTACTACCGAACCCCCCT CTTGCACAGA AATACACGGC AGTGTCGTCA GCCTTTAGGC TGCTGATCTGGAGATATGCC GTGCTGACAG AGGTGTCCAA GGAGAAGGCA AACCGTCCCT TGAAGTCATCAGTATATG-3′

The 3′-terminal sequences (23 nt residues) of hRS7VHA and B arecomplementary to each other. Under defined PCR condition, 3′-ends ofhRS7VHA and B anneal to form a short double stranded DNA flanked by therest of the long oligonucleotides. Each annealed end serves as a primerfor the transcription of the single stranded DNA, resulting in a doublestrand DNA composed of the nt 23 to 340 of hRS7VH. This DNA was furtheramplified in the presence of two short oligonucleotides, hRS7VHBACK (SEQID NO: 21) and hRS7VHFOR (SEQ ID NO: 22) to form the full-length hRS7VH.

(SEQ ID NO: 21) hRS7VHBACK 5′-GTGGTGCTGC AGCAATCTGG GTCTGAGTTGAAGAAGCC-3′ (SEQ ID NO: 22) hRS7VHFOR 5′-TGAGGAGACG GTGACCAGGGACCCTTGGCC CCAGACAT-3′

Minimum amount of hRS7VHA and B (determined empirically) was amplifiedin the presence of 10 μl of 10× PCR Buffer (500 mM KCl, 100 mM Tris.HCLbuffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hRS7VHBACK and hRS7VHFOR, and2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn.).This reaction mixture was subjected to 3 cycle of PCR reactionconsisting of denaturation at 94° C. for 1 minute, annealing at 45° C.for 1 minute, and polymerization at 72° C. for 1.5 minutes, and followedby 27 cycles of PCR reaction consisting of denaturation at 94° C. for 1minute, annealing at 55° C. for 1 minute, and polymerization at 72° C.for 1 minute. Double-stranded PCR-amplified product for hRS7VH wasgel-purified, restriction-digested with PstI and BstEII and cloned intothe complementary PstJBstEII sites of the heavy chain staging vector,VHpBS2.

For constructing the full length DNA of the humanized Vκ sequence,hRS7VKA (156-mer) and hRS7VKB (155-mer) were synthesized as describedabove. hRS7VKA and B were amplified by two short oligonucleotideshRS7VKBACK and hRS7VKFOR as described above.

HRS7VKA (SEQ ID NO: 23) represents nt 20 to 175 of the hRS7Vκ domain.

(SEQ ID NO: 23) 5′-CTCCATCCTC CCTGTCTGCA TCTGTAGGAG ACAGAGTCAGCATCACCTGC AAGGCCAGTC AGGATGTGAG TATTGCTGTA GCCTGGTATC AGCAGAAACCAGGGAAAGCC CCTAAGCTCC TGATCTACTC GGCATCCTAC CGGTACACTG GAGTCC-3′

hRS7VKB (SEQ ID NO: 24) represents the minus strand of the hRS7Vκ domaincomplementary to nt 155 to 320.

(SEQ ID NO: 24) 5′-CCTTGGTCCC AGCACCGAAC GTGAGCGGAG TAATATAATGTTGCTGACAG TAATAAACTG CAAAATCTTC AGGTTGCAGA CTGCTGATGG TGAGAGTGAAATCTGTCCCA GATCCACTGC CACTGAACCT ATCAGGGACT CCAGTGTACC GGTAG-3′ (SEQ IDNO: 25) hRS7VKBACK 5′-GACATTCAGC TGACCCAGTC TCCATCCTCC CTGTCTG-3′ (SEQID NO: 26) hRS7VKFOR 5′-ACGTTAGATC TCCACCTTGG TCCCAGCACC G-3′

Gel-purified PCR products for hRS7Vκ were restriction-digested withPvuII and BglIII and cloned into the complementary PvuI/BcII sites ofthe light chain staging vector, VKpBR2. The final expression vectorhRS7pdHL2 was constructed by sequentially subcloning the XbaI-BamHI andXhoI/BamHI fragments of hRS7Vκ and VH, respectively, into pdHL2 asdescribed above. The full-length cDNA and the encoded amino acidsequences of the light and heavy chains of hRS7 are disclosed in FIG. 5A(SEQ ID NO: 15; SEQ ID NO: 16) and 5B (SEQ ID NO: 17; SEQ ID NO: 18),respectively.

Transfection and Expression of hRS7 Antibodies

Approximately 30 μg of the expression vectors for hRS7 were linearizedby digestion with SalI and transfected into Sp2/0-Ag14 cells byelectroporation (450V and 25 μF). The transfected cells were plated into96-well plates for 2 days and then selected for drug-resistance byadding MTX into the medium at a final concentration of 0.025 μM.MTX-resistant colonies emerged in the wells 2-3 weeks. Supernatants fromcolonies surviving selection were screened for human Ab secretion byELISA assay. Briefly, 100 μl supernatants were added into the wells of amicroliter plate precoated with GAH-IgG, F(ab′)₂ fragment-specific Aband incubated for 1 h at room temperature. Unbound proteins were removedby washing three times with wash buffer (PBS containing 0.05%polysorbate 20). HRP-conjugated GAH-IgG, Fc fragment-specific Ab wasadded to the wells. Following an incubation of 1 h, the plate waswashed. The bound HRP-conjugated Ab was revealed by reading A490 nmafter the addition of a substrate solution containing 4 mM OPD and 0.04%H₂O₂. Positive cell clones were expanded and hRS7 IgG were purified fromcell culture supernatant by affinity chromatography on a Protein Acolumn.

Binding Activity of the Humanized RS7 Antibody

An ELISA competitive binding assay using ME180 cell membrane extractcoated plate was used to assess the immunoreactivity of hRS7 asdescribed (Stein et al., Int. J. Cancer 55:938-946 (1993)). ME180 cellmembrane fraction was prepared by sonication and centrifugation. Thecrude membrane extract was coated in 96-well flat bottomed PVC plate bycentrifugation and fixed with 0.1% glutaraldehyde. Constant amount ofthe biotinylated murine RS7 mixed with varying concentrations of mRS7,cRS7 or hRS7 was added to the membrane coated wells and incubated atroom temperature for 1-2 h. After washing, HRP-conjugated streptavidinwas added and incubated for 1 h at room temperature. The amount ofHRP-conjugated streptavidin bound to the membrane-bound biotinylatedmRS7 was revealed by reading A_(490 nm) after the addition of asubstrate solution containing 4 mM orthophenylenediamine dihydrochlorideand 0.04% H₂O₂. As shown by the competition assays in FIG. 6, hRS7 IgGexhibited comparable binding activities with that of mRS7 and cRS7,confirming the binding affinity of RS7 was preserved in humanization.

EXAMPLE 3 Radioiodinations of Humanized RS7 Using Residualizing Labels

The residualizing moiety (IMP-R4, IMP-R5 or IMP-R8) was radioiodinated,and coupled to disulfide-reduced hRS7 along the procedure describedelsewhere (Govindan S V, et al., Bioconjugate Chem. 1999; 10:231-240).See FIG. 7. In residualizing radioiodine labelings using ¹²⁵I, toprepare ¹²⁵I-IMP-Rx-hRS7 where x=4, 5 or 8), overall yields and specificactivities (in parentheses) of 87.1% (3.38 mCi/mg), 34.3% (0.97 mCi/mg),and 76.6% (2.93 mCi/mg) were obtained using IMP-R4, IMP-R5 and IMP-R8,respectively. In large-scale ¹³¹I labelings using ¹³¹I-IMP-R4 entity,the following results were obtained. Using 20.4 mCi of ¹³¹I, 35.7 nmolof IMP-R4 and 3.22 mg of DTT-reduced hRS7, a 60% overall yield (3.80mCi/mg) was obtained. A different run using 30.3 mCi of ¹³¹I, IMP-R4 andreduced hRS7 produced 69.7% yield (3.88 mCi/mg). A third run using 13.97mCi of ¹³¹I gave 71.8% incorporation (4.42 mCi/mg). A ¹³¹I-IMP-R4labeling using 13.6 mCi of ¹³¹I and a non-specific humanized antibody,hLL2, resulted in 64.4% yield (3.67 mCi/mg).

EXAMPLE 4 Preclinical Experiments in Breast Cancer Animal Model

For tumor targeting studies, tumors were propagated in 5-8 week oldfemale nude mice by subcutaneous injection of ˜2.3×10⁷ culturedMDA-MB-468 cells, and the animals were used after one month when thetumor size reached ˜01-to-0.2 cm³. The mice were injected i.v. with amixture of ˜10 μCi of ¹²⁵I-[IMP-Rx]-hRS7 where x=4, 5 or 8, and 20-25μCi of ¹³¹I-MAb (CT method). Thus, each experiment was a paired-labelexperiment with ¹²⁵I/¹³¹I. At the indicated times, biodistributions invarious organs and blood were determined, and expressed as % injecteddose per gram. Corrections for backscatter of ¹³¹I into ¹²⁵I window weremade in determining ¹²⁵I biodistributions.

For therapy studies, tumor growth patterns under various formats werestudied to determine the optimal method for steady growth of tumor. Itwas concluded that the method used for targeting experiments was optimalafter about 8-weeks of tumor growth, and 30-50% of the animals could beused based on the tumor growth profiles. For therapy studies, thetumor-bearing animals were injected i.v. with ¹³¹I-IMPR4-hRS7 was theagent examined, and compared with directly radioiodinated material,¹³¹I-hRS7. Baseline body weights were compared with weekly measurementsof body weights and tumor volumes. Animals were sacrificed when tumorsreached 3 cm³. All animal experiments were carried out in accord withIACUC-approved protocols.

In Vivo Animal Biodistributions

These experiments were carried out using dual-labeled hRS7 preparations(¹²⁵I-IMP-Rx-hRS7 where x=4, 5 or 8, with each agent mixed with directlabel ¹³¹I-hRS7) in the tumors grown in NIH Swiss nude mice. Tables IA,1B and 1C describe detailed biodistributions showing the superiorperformance using the residualizing labels. For instance, % injecteddose per gram of tumor on day-7 were 41.6±3.0%, 32.2±11.6% and 24.7±8.5%for ¹²⁵I-IMP-R4-hRS7, ¹²⁵I-IMP-R5-hRS7 and ¹²⁵I-IMP-R8-hRS7,respectively, while that for directly labeled ¹³¹I-hRS7 at the sametime-point in each of the dual-labeled experiments were 5.9±0.9%,6.2±2.1% and 6.7±2.3%. Tumor-to-nontumor ratios for the same time-pointwere 1.7-to-7.6-fold higher with ¹²⁵I-IMP-R4-hRS7, 1.7-to-6.0-foldhigher with ¹²⁵I-IMP-R5-S7, and 2.0-to-4.8-fold higher with¹²⁵I-MP-R8-S7 compared to the ratios with ¹³¹I-hRS7 (data not shown).

Table-1. Biodistributions of Humanized RS7, Dual-Labeled with ¹²⁵I-IMP-R(R4 or R5 or R8) and ¹³¹I-hRS7 CT Method in NIH Swiss Nude Mice BearingMDA-MB-468 Tumor Xenografts

TABLE 1A ¹²⁵I-IMP-R4-hRS7 versus ¹³¹I-hRS7 (CT) % ID/g ± SD¹, n = 5Tissue Label 24 h 72 h 168 h, n = 4 336 h MDA-MB-468 ¹²⁵I-IMP-R4 32.8 ±6.3  46.8 ± 11.0 41.6 ± 3.0  25.1 ± 3.8  ¹³¹I (CT) 8.6 ± 1.5 8.6 ± 2.35.9 ± 0.9 4.4 ± 0.8 Tumor wt. (0.19 ± 0.06) (0.19 ± 0.08) (0.13 ± 0.07)(0.18 ± 0.04) Liver ¹²⁵I-IMP-R4 5.7 ± 0.7 4.7 ± 1.5 2.8 ± 0.4 1.3 ± 0.2¹³¹I (CT) 4.1 ± 0.3 2.01 ± 0.1  1.5 ± 0.2 0.7 ± 0.1 Spleen ¹²⁵I-IMP-R43.6 ± 0.6 3.3 ± 0.6 2.6 + 0.8 1.9 ± 0.2 ¹³¹I (CT) 2.6 ± 0.5 1.7 ± 0.41.1 ± 0.4 0.6 ± 0.1 Kidney ¹²⁵I-IMP-R4 7.8 ± 0.7 6.8 ± 0.4 5.6 ± 0.8 3.0± 0.5 ¹³¹I (CT) 3.5 ± 0.3 2.1 ± 0.3 1.4 ± 0.3 0.7 ± 0.1 Lungs¹²⁵I-IMP-R4 4.5 ± 1.0 3.2 ± 0.6 2.2 ± 0.7 0.8 ± 0.2 ¹³¹I (CT) 3.1 ± 0.82.2 ± 0.4 1.6 ± 0.6 0.6 ± 0.2 Blood ¹²⁵I-IMP-R4 15.1 ± 1.4  9.5 ± 0.76.0 ± 1.5 1.9 ± 0.6 ¹³¹I (CT) 10.8 ± 1.0  7.3 ± 0.6 5.3 ± 1.2 2.2 ± 0.6Stomach ¹²⁵I-IMP-R4 1.3 ± 0.2 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I (CT)1.6 ± 0.5 0.7 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 Sm. Int. ¹²⁵I-IMP-R4 1.5 ± 0.20.9 ± 0.1 0.6 ± 0.2 0.2 +± 0.1 ¹³¹I (CT) 1.0 ± 0.1 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.04 Lg. Int. ¹²⁵I-IMP-R4 1.3 ± 0.3 1.0 ± 0.1 0.8 ± 0.1 0.3 ± 0.1¹³¹I (CT) 0.8 ± 0.2 0.5 ± 0.1 0.5 ± 0.1  0.2 ± 0.03 Muscle ¹²⁵I-IMP-R41.2 ± 0.2 0.7 ± 0.1 0.5 ± 0.1 0.3 ± 0.2 ¹³¹I (CT) 0.9 ± 0.1  0.5 ± 0.050.3 ± 0.1 0.2 ± 0.1 Bone ¹²⁵I-IMP-R4 2.3 ± 0.3 2.1 ± 0.3 2.4 ± 0.6 2.3 ±1.2 ¹³¹I (CT) 1.4 ± 0.1 0.8 ± 0.1 0.5 ± 0.1 0.3 ± 0.1

TABLE 1B ¹²⁵I-IMP-R5-hRS7 versus ¹³¹I-hRS7 (CT method) % ID/g ± SD¹, n =5 Tissue Label 24 h 72 h 168 h 336 h, n = 4 MDA-MB-468 ¹²⁵I-IMP-R5 29.1± 4.6  39.6 ± 2.7  32.2 ± 11.6 17.8 ± 7.0  ¹³¹I (CT) 9.2 ± 1.0  9.1 ±_0.6 6.2 ± 2.1 4.9 ± 2.0 Tumor wt. (0.14 ± 0.02) (0.20 ± 0.05) (0.11 ±0.03) (0.13 ± 0.06) Liver ¹²⁵I-IMP-R5 4.8 ± 1.4 2.5 ± 0.1 1.8 ± 0.3 0.8± 0.3 ¹³¹I (CT) 5.1 ± 1.5 2.4 ± 0.2 1.7 ± 0.2 0.8 ± 0.3 Spleen¹²⁵I-IMP-R5 4.1 ± 1.0 2.0 ± 0.4 1.9 ± 0.4 0.8 ± 0.4 ¹³¹I (CT) 3.8 ± 1.21.7 ± 0.5 1.3 ± 0.3 0.7 ± 0.4 Kidney ¹²⁵I-IMP-R5 10.0 ± 1.4  6.3 ± 0.55.0 ± 0.5 1.1 ± 0.3 ¹³¹I (CT) 3.7 ± 0.5 1.9 ± 0.3 1.7 ± 0.3 0.8 ± 0.2Lungs ¹²⁵I-IMP-R5 5.4 ± 1.8 3.2 ± 0.8 2.3 ± 0.2 0.9 ± 0.4 ¹³¹I (CT) 3.9± 1.2 2.5 ± 0.7 2.0 ± 0.3 0.9 ± 0.5 Blood ¹²⁵I-IMP-R5 16.5 ± 4.0  8.8 ±0.6 6.5 ± 1.0 2.7 ± 1.4 ¹³¹I (CT) 12.2 ± 3.0  7.8 ± 0.5 6.3 ± 0.8 3.1 ±1.4 Stomach ¹²⁵I-IMP-R5 0.9 ± 0.2 0.5 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I(CT) 1.1 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 0.2 ± 0.1 Sm. Int. ¹²⁵1-IMP-R5 1.5 ±0.3  0.8 ± 0.04 0.6 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 1.1 ± 0.2  0.6 ± 0.02 0.5± 0.1 0.3 ± 0.1 Lg. Int. ¹²⁵I-IMP-R5 1.4 ± 0.2 0.9 ± 0.1 0.6 ± 0.1  0.2± 0.04 ¹³¹I (CT) 0.7 ± 0.1  1.4 ± 0.03 0.4 ± 0.1  0.2 ± 0.04 Muscle¹²⁵I-IMP-R5 1.3 ± 0.3 0.7 ± 0.2 0.5 ± 0.1 0.2 ± 0.1 ¹³¹I(CT) 0.9 ± 0.20.6 ± 0.2 0.4 ± 0.1 0.2 ± 0.1 Bone ¹²⁵I-IMP-R5 2.2 ± 0.6 1.3 ± 0.2 1.2 ±0.5 1.0 ± 0.6 ¹³¹I (CT) 1.9 ± 0.7 0.9 ± 0.1 0.6 ± 0.2 0.3 ± 0.2

TABLE 1C ¹²⁵I-IMP-R8-hRS7 versus ¹³¹I-hRS7 (CT method) % ID/g ± SD¹, n =5 Tissue Label 24 h 72 h 168 h 336 h MDA-MB-468 ¹²⁵I-IMP-R8 29.1 ± 5.4 29.6 ± 3.9  24.7 ± 8.5  11.0 ± 6.4  ¹³¹I (CT) 8.8 ± 1.6 8.8 ± 1.0 6.7 ±2.3 2.4 ± 1.3 Tumor wt. (0.17 ± 0.04) (0.12 ± 0.05) (0.10 ± 0.04) (0.15± 0.05) Liver ¹²⁵I-IMP-R8 4.6 ± 0.7 3.3 ± 0.4 1.8 ± 0.2 0.7 ± 0.2 ¹³¹I(CT) 4.1 ± 0.6 3.3 ± 0.4 1.8 ± 0.2 0.8 ± 0.2 Spleen ¹²⁵I-IMP-R8 2.6 ±0.7 2.3 ± 0.2 1.9 ± 0.2 1.0 ± 0.1 ¹³¹I (CT) 2.4 ± 0.8 2.2 ± 0.3 2.0 ±0.3 0.7 ± 0.1 Kidney ¹²⁵I-IMP-R8 7.2 ± 0.8 4.6 ± 0.8 2.6 ± 1.0 1.8 ± 0.1¹³¹I (CT) 2.5 ± 0.3 3.0 ± 0.7 1.8 ± 0.5 0.8 ± 0.3 Lungs ¹²⁵I-IMP-R8 3.0± 0.7 4.7 ± 0.5 2.3 ± 0.6 1.0 ± 0.4 ¹³¹I (CT) 2.4 ± 0.4 4.4 ± 0.5 2.1 ±0.5 1.0 ± 0.4 Blood ¹²⁵I-IMP-R8 10.8 ± 1.2  9.6 ± 0.9 6.3 ± 1.4 2.2 ±0.6 ¹³¹I (CT) 9.2 ± 1.6 9.5 ± 0.8 6.4 ± 1.4 2.6 ± 0.6 Stomach¹²⁵I-IMP-R8 0.9 ± 0.2 0.7 ± 0.2 0.3 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 1.1 ± 0.20.9 ± 0.3 0.4 ± 0.1 0.3 ± 0.1 Sm. Int. ¹²⁵I-IMP-R8 1.0 ± 0.1 0.8 ± 0.20.5 ± 0.1 0.2 ± 0.1 ¹³¹I (CT) 0.8 ± 0.1 0.8 ± 0.1 0.5 ± 0.1 0.2 ± 0.1Lg. Int. ¹²⁵I-IMP-R8 1.0 ± 0.1 0.9 ± 0.1 0.5 ± 0.1 0.3 ± 0.1 ¹³¹I (CT)0.6 ± 0.1 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 Muscle ¹²⁵I-IMP-R8 0.8 ± 0.1 0.6± 0.1 0.4 ± 0.1 0.2 ± 0.1 ¹³¹I(CT)  0.6 ± 0.04 0.6 ± 0.1 0.4 ± 0.1 0.2 ±0.1 Bone ¹²⁵I-IMP-R8 1.4 ± 0.2 1.2 ± 0.3 1.4 ± 0.2 0.8 ± 0.2 ¹³¹I (CT)1.1 ± 0.2 0.9 ± 0.2 0.7 ± 0.1 0.3 ± 0.1

Dosimetry calculations, based on biodistributions using ¹²⁵I in place of¹³¹I, were performed using the method of Siegel, J A and Stabin, M G(Journal of Nuclear Medicine, 1994; 35:152-156). Table-2 compares setsof residualizing and conventional radioiodine labels, and FIG. 8describes the data graphically. All of the residualizing agents are seento perform optimally in terms of dose delivered to tumor andtumor-to-nontumor ratios; ¹³¹I-IMP-R4-hRS7 was chosen for therapyexperiments in view of the advantageous radiochemical yields andspecific activities obtainable for the same agent.

TABLE 2 Calculated radiation doses due to variously radioiodinated hRS7in the MDA-MB-468 tumor model cGy normalized to 1500 cGy to Blood GroupI Group II Group III Organ Model IMP-R4 CT IMP-R5 CT IMP-R8 CT Tumor(Trap 0 6995 1613 5187 1506 4000 1206 point 0) Liver Exp 674 456 398 449497 505 Spleen Exp 535 315 336 313 384 356 Kidney Exp 1063 402 867 361761 394 Lungs Exp 450 392 450 422 506 473 Blood (org) Exp 1500 1500 15001500 1500 1500 Stomach Exp 104 144 84 118 101 128 Sm Int Exp 148 124 131119 130 121 Lg Int Exp 163 108 136 86 140 97 Muscle Exp 112 99 105 10097 93 Bone Exp 486 151 244 149 245 151 mCi for 0.231 0.285 0.213 0.2390.248 0.255 1500 cGy to bloodTherapy of MDA-MB-468 Human Breast Carcinoma Xenografts in Nude Mice

Maximum-tolerated-dose (MTD): From dosimetry data (Table-2, group-1),the mCi amounts of ¹³¹I-IMP-R4-hRS7 and ¹³¹I-hRS7, producing a radiationdose of 1500 cGy to blood (estimated MTD) were calculated to be 0.231mCi and 0.285 mCi, respectively. Experimental determination of MTD wascarried out using increasing doses of each agent in Swiss nude mice. For¹³¹I-IMP-R4-hRS7, groups of animals were administered 200, 225, 250,275, 300 and 325 μCi; 1 out of five animals in the 250 μCi dose groupdied by week 4, while 3 out of 4 animals in the 300 μCi dose group diedbetween weeks 2 and 4. Although the survival of animals in the 275 and325 μCi dose groups at five weeks was unexpected, we concluded that theMTD was between 231 μCi (calculated from dosimetry data) and 250 μCi ofadministered dose. For the ¹³¹I-hRS7 (‘CT’-based radioiodination),groups of animals were injected with 250, 280, 310, 340, 370 and 400μCi; between weeks 2 and 3, six out of six animals of 340 μCi dosegroup, three out of six animals of 370 μCi dose group, and four out offour animals of 400 μCi dose group died. Based on these, the MTD wasprojected to be in the 280-310 μCi range.

Therapy Study-1

For this first therapy experiment, comparing the efficacy of¹³¹I-IMPR-4-hRS7 with that of ¹³¹I-hRS7 (CT method), each agent used at˜70% of its maximum-tolerated dose. A single dose of 175 μCi of theresidualizing agent is seen to be significantly more effective than 200μCi of conventional radioiodine agent. In this experiment, which alsoincluded untreated controls, 10 or 11 animals were used per group, andall the three groups were randomized such that the distribution ofstarting tumor sizes were very similar. Mean tumor volumes for the threegroups before therapy (day-2) were 0.312±0.181, 0.308±0.203, and0.303±0.212.

In this experiment, interim data to day 49 are depicted in FIG. 9 below.The top panel in FIG. 9 shows tumor volumes (cm³) for individual animalsin each group, and the bottom panel indicates mean tumor volumes in twoformats. There were three deaths in the untreated group. Tumor growthcontrol is significantly better for the residualizing label groupcompared to the conventional label and the untreated groups, asdetermined by the student-t test on the area under the curves (AUC) formean tumor volumes (MTV) up to day-49. On day 49, significance (pvalues) for differences in AUCs of MTVs due to therapy with¹³¹I-IMP-R4-hRS7, with the respective p values for tumor volumedifferences before therapy (day-2) given in parentheses, are as follows.Versus untreated: 0.05 (0.78); versus ¹³¹I-hRS7 (CT): 0.03 (0.98); for-hRS7 (CT) versus untreated: 0.14 (0.81). There is continuing divergencein mean tumor volumes between the conventional and the residualizingradioiodine groups on day 49, with the latter group leading to continueddecrease. At 8-weeks post-therapy, complete remissions were observed in5 of 11 mice treated with ¹³¹I-IMP-R4-hRS7, and the MTV was 20% of thestarting value. MTV in the untreated and ¹³¹I-hRS7-treated mice at 8weeks were 280% and 163% of the respective starting values,respectively, with 1 complete remission of 11 mice in the ¹³¹I-hRS7group.

The treatments were well tolerated. The mean body weights of IMP-R4group on day-2 was 21.93±2.03 and that on day 49 was 23.68±1.81; for‘CT’ group, the mean body weights were 21.77±2.21 and 23.90±2.64 ondays-2 and 49, respectively. Myelotoxicities of the treated groups, asdetermined by blood cell counts, are shown in FIG. 10. Briefly: With¹³¹I-IMP-R4-hRS7, nadirs of 34%, 7% and 61% of the control levels forWBC, lymphocite and neutrophil counts, respectively, were reached oneweek after the administration of the agent. By week-5, these recoveredto 74%, 58% and 92% of the control levels, respectively, and remained at45%, 36% and 51% of the control levels on day-49; and for ¹³¹I-hRS7(CT): nadirs of 41%, 13% and 67% of the control levels for WBC,lymphocite and neutrophil counts, respectively, were reached one weekafter the administration of the agent. By week-5, these recovered to85%, 67% and 103% of the control levels, respectively, and remained at42%, 32% and 49% of the control levels on day-49.

Therapy Study-2

Specificity of RAIT Using ¹³¹I-IMP-R4-hRS7 in the MDA-MB-468 Tumor Model

The efficacy of ¹³¹I-IMP-R4-hRS7 was compared with that of non-specificcontrol humanized antibody, hLL2 (anti-CD-22 MAb), labeled with¹³¹I-IMP-R4. In this experiment, 175 μCi of each agent was administered.This represents ˜70% of the maximum-tolerated dose of ¹³¹I-IMP-R4-hRS7.In this experiment, which included untreated controls, 7-to-8 animalswere used per group, and the groups were randomized with regard to thestarting tumor volume distributions as in therapy experiment-1. FIG. 11,showing the relative mean tumor volumes (MTV) for the three groups (MTVbefore therapy: 100), is indicative of the growth control specificity.

EXAMPLE 5 Treatment of a Breast Cancer Patient with Y-90 Humanized RS7mAb and with Naked Humanized RS7 mAb

A 56-year-old women with a history of recurrent adencarcinoma of thebreast presents with cervical lymph node and left lung metastases. Sherelapses twice after chemotherapy and hormonal therapies. She is thengiven two therapeutic injections, two weeks apart, of Y-90-conjugatedhumanized RS7 mAb i.v., at a dose each of 20 mCi Y-90 in a protein doseof antibody of 100 mg. Four weeks after therapy, her white blood celland platelet counts have decreased by approximately 50%, but recuperateby 9 weeks post-therapy. At the restaging 12 weeks post-therapy, a ca.30% decrease in pulmonary and nodal metastases has been measured bycomputed tomography. Thereafter, she receives 4 weekly infusions, over 3hours each, of naked humanized RS7, which is tolerated well, except forsome transient rigors and chills, and without any adverse effects on herblood counts or blood chemistries. The naked antibody dose for eachinfusion was 400 mg/m². Approximately 8 weeks later, restaging bycomputed tomography indicates an additional decrease in measurablelesions by about 20 percent. At the followup examination 3 months later,her disease appears to be stable (i.e., no evidence of additional, orprogressive growth).

1. An isolated humanized antibody or fragment thereof that competes forbinding to EGP-1 (TROP2) glycoprotein with a murine antibody comprisinglight chain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:28); CDR2 (SASYRYT, SEQ ID NO:29); and CDR3(QQHYITPLT, SEQ ID NO:30) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:31); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:32) and CDR3(GGFGSSYWYFDV, SEQ ID NO:33), wherein said humanized antibody compriseshuman antibody framework region (FR) sequences and said FR sequencescomprise at least one amino acid residue substituted by an amino acidresidue selected from the group consisting of amino acid residues 38,46, 68 and 91 of the murine antibody heavy chain variable region of SEQID NO:4 and residues 20, 85 and 100 of the murine antibody light chainvariable region of SEQ ID NO:2.
 2. The humanized antibody or fragmentthereof of claim 1, comprising light chain framework region (FR)sequences of the human SA-1A'cl antibody (SEQ ID NO:5), heavy chainFR1-F3 sequences of the human RF-TS3 antibody (SEQ ID NO:8) and heavychain FR4 sequence of the human NEWM antibody (SEQ ID NO:6).
 3. Thehumanized antibody or fragment thereof of claim 1, wherein the FR aminoacid substitutions comprise the amino acid residues 38, 46, 68 and 91 ofthe murine antibody heavy chain variable region of SEQ ID NO:4 andresidues 20, 85 and 100 of the murine antibody light chain variableregion of SEQ ID NO:2.
 4. The humanized antibody or fragment thereof ofclaim 1, wherein the antibody or fragment comprises the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:28); CDR2 (SASYRYT, SEQ IDNO:29); and CDR3 (QQHYITPLT, SEQ ID NO:30) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:31); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:32) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:33).
 5. The isolated humanizedantibody or fragment thereof of claim 1, wherein said antibody orfragment is a naked antibody or fragment.
 6. The isolated humanizedantibody or fragment thereof of claim 1, wherein said antibody orfragment is conjugated to at least one diagnostic or therapeutic agent.7. The isolated humanized antibody or fragment thereof of claim 6,wherein the diagnostic or therapeutic agent is selected from the groupconsisting of a drug, a toxin, an immunomodulator, a hormone, an enzyme,a growth factor, a radionuclide, an antisense oligonucleotide, a metal,a dye, a fluorescent agent, a chemiluminescent agent, a bioluminescentagent, a paramagnetic ion and a contrast agent.
 8. The isolatedhumanized antibody or fragment thereof of claim 7, wherein the toxin isselected from the group consisting of Pseudomonas exotoxin, ricin,abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin and Pseudomonasexotoxin.
 9. The isolated humanized antibody or fragment thereof ofclaim 7, wherein the immunomodulator is selected from the groupconsisting of a cytokine, stem cell growth factor, lymphotoxin, tumornecrosis factor (TNF), hematopoietic factor, interleukin-1 (IL-1), IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, colony stimulating factor,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, the stem cell growth factor designated “S1factor,” erythropoietin and thrombopoietin.
 10. The isolated humanizedantibody or fragment thereof of claim 7, wherein the radionuclide isselected from the group consisting of ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu,⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ⁹⁰Y, ¹¹¹In, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,¹⁷⁷Lu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ²¹¹At, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br,^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(125m)Te,¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Ag, ¹¹¹Ag, ¹⁹⁷Pt, ¹⁰⁹Pd, ³²P, ³³P, ⁴⁷Sc, ¹⁵³Sm, ¹⁰⁵Rh,¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ¹⁸F, ⁷⁵Se,²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ⁸⁶Y, ¹⁶⁹Yb, ¹⁶⁶Dy, ²¹²Pb, and ²²³Ra.
 11. Theisolated humanized antibody or fragment thereof of claim 7, wherein thetherapeutic agent has a pharmaceutical property selected from the groupconsisting of antimitotic, alkylating, antimetabolite, antiangiogenic,apoptotic, alkaloid and antibiotic.
 12. The isolated humanized antibodyor fragment thereof of claim 7, wherein the therapeutic agent isselected from the group consisting of a nitrogen mustard, ethyleniminederivative, alkyl sulfonate, nitrosourea, triazene, folic acid analog,anthracycline, taxane, COX-2 inhibitor, tyrosine kinase inhibitor,pyrimidine analog, purine analog, antibiotic, enzyme,epipodophyllotoxin, platinum coordination complex, vinca alkaloid,substituted urea, methyl hydrazine derivative, adrenocorticalsuppressant, endostatin, taxol, camptothecin, doxorubicin anddoxorubicin analog.
 13. The isolated humanized antibody or fragmentthereof of claim 1, wherein the fragment is an F(ab′)₂, Fab′, F(ab)₂,Fab, Fv, sFv or scFv fragment.
 14. The isolated humanized antibody orfragment thereof of claim 1, wherein the antibody or fragment forms partof a fusion protein.
 15. The isolated humanized antibody or fragmentthereof of claim 14, wherein the fusion protein is conjugated to atleast one diagnostic or at least one therapeutic agent.
 16. Acomposition for treating cancer comprising at least one naked humanizedantibody or fragment thereof that competes for binding to EGP-1 (TROP2)glycoprotein with a murine antibody comprising light chaincomplementarity-determining region (CDR) sequences CDR1 (KASQDVSIAVA,SEQ ID NO:28); CDR2 (SASYRYT, SEQ ID NO:29); and CDR3 (QQHYITPLT, SEQ IDNO:30) and heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:31); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:32) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:33).17. The composition of claim 16, wherein the antibody is a humanizedantibody comprising the light chain framework region (FR) sequences ofthe human SA-1A'cl antibody (SEQ ID NO:5), heavy chain FR1F-3 sequencesof the human RF-TS3 antibody (SEQ ID NO:8) and heavy chain FR4 sequenceof the human NEWM antibody (SEQ ID NO:6).
 18. The composition of claim17, wherein said FR sequences comprise at least one amino acid residuesubstituted by an amino acid residue selected from the group consistingof amino acid residues 38, 46, 68 and 91 of the murine antibody heavychain variable region of SEQ ID NO:4 and residues 20, 85 and 100 of themurine antibody light chain variable region of SEQ ID NO:2.
 19. Thecomposition of claim 18, wherein the FR amino acid substitutionscomprise the amino acid residues 38, 46, 68 and 91 of the murineantibody heavy chain variable region of SEQ ID NO:4 and residues 20, 85and 100 of the murine antibody light chain variable region of SEQ IDNO:2.
 20. The composition of claim 16, further comprising at least onetherapeutic agent selected from the group consisting of a drug, a toxin,an immunomodulator, a hormone, an enzyme, a second antibody or fragmentthereof and an immunoconjugate.
 21. The composition of claim 16, whereinthe humanized antibody or fragment thereof comprises the light chaincomplementarity-determining region (CDR) sequences CDR1 (KASQDVSIAVA,SEQ ID NO:28); CDR2 (SASYRYT, SEQ ID NO:29); and CDR3 (QQHYITPLT, SEQ IDNO:30) and heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:31); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:32) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:33).22. The composition of claim 20, wherein the toxin is selected from thegroup consisting of Pseudomonas exotoxin, ricin, abrin, ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtheria toxin and Pseudomonas exotoxin.
 23. Thecomposition of claim 20, wherein the immunomodulator is selected fromthe group consisting of a cytokine, stem cell growth factor,lymphotoxin, tumor necrosis factor (TNF), hematopoietic factor,interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21,colony stimulating factor, granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF),interferon-α, interferon-β, interferon-γ, the stem cell growth factordesignated “S1 factor,” erythropoietin and thrombopoietin.
 24. Thecomposition of claim 20, wherein the therapeutic agent has apharmaceutical property selected from the group consisting ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid and antibiotic.
 25. The composition of claim 20, wherein thetherapeutic agent is selected from the group consisting of a nitrogenmustard, ethylenimine derivative, alkyl sulfonate, nitrosourea,triazene, folic acid analog, anthracycline, taxane, COX-2 inhibitor,tyrosine kinase inhibitor, pyrimidine analog, purine analog, antibiotic,enzyme, epipodophyllotoxin, platinum coordination complex, vincaalkaloid, substituted urea, methyl hydrazine derivative, adrenocorticalsuppressant, endostatin, taxol, camptothecin, doxorubicin anddoxorubicin analog.
 26. The composition of claim 20, wherein thefragment is an F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv or scFv fragment. 27.The composition of claim 20, wherein the antibody or fragment forms partof a fusion protein.
 28. The composition of claim 20, wherein the secondantibody binds to an antigen selected from the group consisting of CD20,carcinoembryonic antigen, CSAp, alpha-fetoprotein, PAM4 antigen,EGP-1(TROP2), EGP-2, MUC-1, MUC-2, MUC-3, MUC-4, CC49, PSMA, PSA, EGFR,A33, HER2/neu, Le(y), Tn, Tag-72, HCG, HCG-beta, ferritin, PAP, EGP-2,tenascin, CanAg, G250, VGFR1 and VGFR2.